Search Images Maps Play YouTube News Gmail Drive More »
Sign in
Screen reader users: click this link for accessible mode. Accessible mode has the same essential features but works better with your reader.

Patents

  1. Advanced Patent Search
Publication numberUS6410323 B1
Publication typeGrant
Application numberUS 09/387,341
Publication dateJun 25, 2002
Filing dateAug 31, 1999
Priority dateAug 31, 1999
Fee statusLapsed
Also published asUS20030199467, WO2001015739A1
Publication number09387341, 387341, US 6410323 B1, US 6410323B1, US-B1-6410323, US6410323 B1, US6410323B1
InventorsM. Luisa Roberts, Lex M. Cowsert
Original AssigneeIsis Pharmaceuticals, Inc.
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Nucleotide sequences for use in the treatment of cell proliferation defects
US 6410323 B1
Abstract
This invention provides compositions and methods for modulating expression of members of the human Rho gene family, which encode low molecular weight GTPases that act as molecular switches in signal transduction. In preferred embodiments, Rho family members include RhoA, RhoB, RhoC, RhoG, Rac1 and cdc42. This invention is also directed to methods for inhibiting hyperproliferation of cells; these methods can be used diagnostically or therapeutically. Furthermore, this invention is directed to treatment of conditions associated with expression of the human Rho family members, particularly in hyperproliferative disorders.
Images(65)
Previous page
Next page
Claims(13)
What is claimed is:
1. An antisense compound 8 to 30 nucleobases in length targeted to an nucleic acid molecule encoding human cdc42 that comprises at least an 8 nucleobase portion of SEQ ID NO: 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229 or 230, and wherein said antisense compound inhibits expression of human cdc42.
2. The antisense compound of claim 1, which is an antisense oligonucleotide.
3. The antisense compound of claim 2, wherein the oligonucleotide comprises at least one modified internucleoside linkage.
4. The antisense compound of claim 3, wherein the modified internucleoside linkage is a phosphorothioate linkage.
5. The antisense compound of claim 2 wherein the oligonucleotide comprises at least one modified sugar moiety.
6. The antisense compound of claim 5 wherein the modified sugar moiety is a 2′-O-methoxyethyl sugar moiety.
7. The antisense compound of claim 2 wherein the oligonucleotide comprises at least one modified nucleobase.
8. The antisense compound of claim 7 wherein the modified nucleobase is a 5-methylcytosine.
9. The antisense compound of claim 2, wherein the oligonucleotide is a chimeric oligonucleotide.
10. A composition comprising the antisense compound of claim 2, and a pharmaceutically acceptable carrier or diluent.
11. The composition of claim 10, further comprising a colloidal dispersion system.
12. The composition of claim 2, wherein the antisense compound is an antisense oligonucleotide.
13. A method of inhibiting the expression of human cdc42 in human cells and tissues comprising contacting said cells or tissues in vitro with the antisense compound of claim 1, is so that expression of said human cdc42 is inhibited.
Description
FIELD OF THE INVENTION

This invention relates to compositions and methods for modulating expression of members of the human Rho gene family, which encode low molecular weight GTPases that act as molecular switches in signal transduction. This invention is also directed to methods for inhibiting hyperproliferation of cells; these methods can be used diagnostically or therapeutically. Furthermore, this invention is directed to treatment of conditions associated with expression of the human Rho family member genes.

BACKGROUND OF THE INVENTION

The Rho family of genes are a sub-family of low molecular weight GTPases and are related to each other based on sequence homology and function (Vojtek, A. B., and Cooper, J. A., Cell 1995, 82, 527-529). Other sub-families include Ras, Rab, Arf, and Ran. As GTPases, these proteins bind and hydrolyze GTP. In an active state, they bind to GTP and transduce signals of other proteins in signal transduction pathways. In their inactive state, they are bound to GDP. Members of the Rho family are typically involved in regulation of the actin cytoskeleton. Members of the Rho family include RhoA, RhoB, RhoC, RhoD, RhoE, RhoG, Rac1, Rac2, Rac3 and Cdc42.

Each class appears to have a unique function in actin reorganization. Rho has been shown to be essential for the formation of stress fibers and focal adhesions (Ridley, A. J. and Hall, A., Cell 1992, 70, 389-399). Focal adhesions are an area of the cell where integrin receptors cluster and extracellular matrix proteins such as fibronectin and collagen are bound. Stress fibers attach at these focal adhesions within a cell. Rac has been shown to be essential for the formation of membrane ruffles, which results from the formation of large vesicles within the cell (Ridley, A. J., et al., Cell 1992, 70, 401-410). Cdc42 (also known as Cdc42Hs and G25K) regulates the formation of filopodia, short bundles of actin filaments that protrude from a cell (Nobes, C. D. and Hall, A., Cell 1995, 81, 53-62). Such activities on cell morphology may play an important role in cell motility, cytokinesis, and endocytosis.

Additional functions for the Rho family have begun to be elucidated. Rac and Rho have been found to promote cadherin-based cell-cell adhesion (Takaishi, K., et al., J. Cell Biol. 1997, 139, 1047-1059). Rac1 and Cdc42 play a critical role in the c-jun amino-terminal kinase (JNK)/stress-activated protein kinase (SAPK) signaling pathway, thereby, potentially having an important role in gene transcription (Coso, O. A. et al., Cell 1995, 81, 1137-1146). RhoA, Rac1 and Cdc42 also regulate transcription through JNK-independent pathways by binding to either serum response factor (SRF; Hill, C. S., et al., Cell 1995, 81, 1159-1170) or NF-κB (Perona, R., et al., Genes and Develop. 1997, 11, 463-475).

Members of the Rac subfamily have also been found to regulate oxygen radical production. Both Rac1 (Sundaresan, M., et al., Biochem. J. 1996, 318, 379-382) and Rac2 (Knaus, U. G., et al., Science 1991, 254, 1512-1515) are involved in this process.

Members of the Rho family are thought to be involved in various disease processes, including cancer. Rho, Rac and Cdc42 all play a role in Ras transformation. Rac was found to essential for transformation by Ras, but not RafCAAX, a modified Raf kinase with a localization signal from K-ras (Qiu, R.-G., et al., Nature 1995 374, 457-459). Rho is not essential for Ras transformation, but acts cooperatively in transformation by Ras and RafCAAX (Qiu, R.-G., et al., Proc. Natl. Acad. Sci. USA 1995, 92, 11781-11785). Cdc42 was also found to be essential for Ras transformation, but its role is distinct from that of Rac (Qiu, R.-G., et al., Mol. Cell Biol. 1997, 17, 3449-3458). In addition to transformation, members to of the Rho family may also play a role in invasion and metastasis. Michiels, F. et al. (Nature 1995, 375, 338-340) demonstrated that T-lymphoma cells that constitutively expressed Rac1 became invasive. Yoshioka, K. et al. (J. Biol. Chem. 1998, 273, 5146-5154) found that cells stably transfected with RhoA were also invasive. The RhoB gene has been classified as an immediate-early gene, which means that its transcription is rapidly activated upon exposure to certain growth factors or mitogens. The factors shown to activate RhoD transcription include epidermal growth factor (EGF), platelet-derived growth factor (PDGF), genotoxic stress from UV light, alkylating xenobiotics and the retroviral oncogene v-fps. Each of these stimuli triggers DNA synthesis in cultures of high cell density (Engel et al., J. Biol. Chem., 1998, 273, 9921-9926). The response of RhoB to these factors implies a role for RhoB in wound repair and tissue regeneration upon growth factor stimulation and tumorigenesis upon mitogen stimulation.

The involvement of Rho family proteins in ras-mediated transformation and tumor cell invasion suggests that they could be novel targets for cancer treatment (Ridley, A. J., Int. J. Biochem. Cell Biol. 1997, 29, 1225-1229). In particular, overexpression of the RhoC gene has been associated with pancreatic cancer. Suwa, H. et al. (Br. J. Cancer, 1998, 77, 147-152) looked for a role of RhoA, RhoB and RhoC genes in ductal adenocarcinoma of the pancreas. They found that expression levels of RhoC were higher in tumors than in normal tissue and that metastatic tumors expressed RhoC at higher levels than primary tumors. Rho C expression is also elevated in a megakaryocytic leukemia cell line, CMK. Takada et al., Exp. Hematol., 1996, 24, 524-530. Manifestations of altered RhoB regulation also appear in disease states, including the development of cancer. Cellular transformation and acquisition of the metastatic phenotype are the two main changes normal cells undergo during the progression to cancer. Expression of constitutively activated forms of RhoB have been shown to cause tumorigenic transformation of NIH 3T3 and Rat1 rodent fibroblasts (Khosravi-Far et al., Adv. Cancer Res., 1998, 72, 57-107). RhoB has also been shown to be overexpressed in human breast cancer tissues (Zalcman et al., Oncogene, 1995, 10, 1935-1945). RhoA is also believed to be involved in the development of cancer. Cellular transformation and acquisition of the metastatic phenotype are the two main changes normal cells undergo during the progression to cancer. Recent studies demonstrate that RhoA-regulated pathways can induce both changes in cells. Injecting cells transformed with rhoA genes directly into the bloodstream of mice produced metastasis, or tumor growth, in distant organs (del Peso et al., Oncogene, 1997, 15, 3047-3057).

It has also been suggested that inhibition of Rac genes may be useful for preventing reoxygenation injury as it occurs when ischemic cells undergo reperfusion (Kim, K.-S., et al., J. Clin. Invest. 1998, 101, 1821-826). With reoxygenation, reactive oxygen species are presented to the cell, greatly augmenting cell death. Kim, K.-S., et al. showed that adenoviral-mediated transfer of a dominant negative Rac1 could inhibit the formation of reactive oxygen species and protect cells against hypoxia/reoxygenation-induced cell death. They suggest that inhibition of rac1 would be useful, clinically, in treatment in cases where there is the possibility of reperfusion injury.

Manifestations of altered RhoA regulation also appear in both injury and disease states. It has been proposed that acute central nervous system trauma may contribute to the development of Alzheimer's disease. Findings that show a high concentration of thrombin, a serine-protease in the blood clotting cascade, localized to the plaques of Alzheimer's disease brains support this claim. An excess of thrombin has been shown to stimulate Rho A activity with a concomitant increase in apoptosis (programmed cell death) (Donovan et al., J. Neurosci., 1997, 17, 5316-5326). These studies also imply a role for RhoA in wound repair and clotting disorders.

Although members of the Rho family have been implicated in various disease processes including cancer and reoxygenation injury, no effective therapy specifically targeting these proteins is available. Antisense oligonucleotides have been used to study the role of some Rho family members in various physiological processes. Dorseuil, O., et al. (J. Biol. Chem. 1992, 267, 20540-20542) used an 16-mer antisense oligonucleotide targeted to the start site of both Rac1 and Rac2 and demonstrated a dose-dependent reduction in superoxide production in whole cells. Brenner, B., et al. (Biochem. Biophys. Res. Commun. 1997, 231, 802-807) used a similar oligonucleotide (a 15-mer targeted to the start site) and showed that inhibition of Rac2 protein expression prevented L-selectin-induced actin polymerization. An 45-mer antisense oligonucleotide targeted to the 3′-UTR has also been used as a probe for rac1 (Didsbury, I., et al., J. Biol. Chem. 1989, 264, 16378-16382).

Thus, there remains an unmet need for compositions and methods targeting expression of Rho family members, and disease processes associated there-with.

SUMMARY OF THE INVENTION

The present invention provides oligonucleotides which are targeted to nucleic acids encoding members of the human Rho gene family and are capable of modulating Rho family members expression. The present invention also provides chimeric oligonucleotides targeted to nucleic acids encoding human Rho family members. The oligonucleotides of the invention are believed to be useful both diagnostically and therapeutically, and are believed to be particularly useful in the methods of the present invention.

The present invention also comprises methods of modulating the expression of human Rho family members using the oligonucleotides of the invention. Methods of inhibiting Rho family members expression are provided; these methods are believed to be useful both therapeutically and diagnostically. These methods are also useful as tools, for example, for detecting and determining the role of Rho family member expression in various cell functions and physiological processes and conditions and for diagnosing conditions associated with expression of Rho family members.

The present invention also comprises methods for diagnosing and treating cancer and preventing reoxygenation injury. These methods are believed to be useful, for example, in diagnosing Rho family member-associated disease progression. These methods employ the oligonucleotides of the invention. These methods are believed to be useful both therapeutically, including prophylactically, and as clinical research and diagnostic tools.

DETAILED DESCRIPTION OF THE INVENTION

Members of the Rho family of GTPases are essential for transformation by Ras and play a role in tumor cell invasion. In addition, the Rac subfamily is a regulator of oxygen radical formation. As such, they represent attractive targets for antineoplastic therapy and preventative agents for radical deoxygenation. In particular, modulation of the expression of RhoC may be useful for the treatment of pancreatic carcinomas and modulation of Rac1 may be useful for preventing ischemia/reperfusion injury.

Antisense oligonucleotides targeting members of the Rho family represent a novel therapeutic approach.

The present invention employs antisense compounds, particularly oligonucleotides, for use in modulating the function of nucleic acid molecules encoding Rho family members, ultimately modulating the amount of a Rho family member produced. This is accomplished by providing oligonucleotides which specifically hybridize with nucleic acids, preferably mRNA, encoding a Rho family member.

This relationship between an antisense compound such as an oligonucleotide and its complementary nucleic acid target, to which it hybridizes, is commonly referred to as “antisense”. “Targeting” an oligonucleotide to a chosen nucleic acid target, in the context of this invention, is a multistep process. The process usually begins with identifying a nucleic acid sequence whose function is to be modulated. This may be, as examples, a cellular gene (or mRNA made from the gene) whose expression is associated with a particular disease state, or a foreign nucleic acid from an infectious agent. In the present invention, the targets are nucleic acids encoding Rho family members; in other words, a gene encoding a Rho family member, or mRNA expressed from a Rho family member gene. mRNA which encodes a Rho family member is presently the preferred target. The targeting process also includes determination of a site or sites within the nucleic acid sequence for the antisense interaction to occur such that modulation of gene expression will result.

In accordance with this invention, persons of ordinary skill in the art will understand that messenger RNA includes not only the information to encode a protein using the three letter genetic code, but also associated ribonucleotides which form a region known to such persons as the 5′-untranslated region, the 3′-untranslated region, the 5′ cap region and intron/exon junction ribonucleotides. Thus, oligonucleotides may be formulated in accordance with this invention which are targeted wholly or in part to these associated ribonucleotides as well as to the informational ribonucleotides. The oligonucleotide may therefore be specifically hybridizable with a transcription initiation site region, a translation initiation codon region, a 5′ cap region, an intron/exon junction, coding sequences, a translation termination codon region or sequences in the 5′- or 3′-untranslated region. Since, as is known in the art, the translation initiation codon is typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the “AUG codon,” the “start codon” or the “AUG start codon.” A minority of genes have a translation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, the terms “translation initiation codon” and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, “start codon” and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding a Rho family member, regardless of the sequence(s) of such codons. It is also known in the art that a translation termination codon (or “stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively). The terms “start codon region,” “AUG region” and “translation initiation codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation initiation codon. This region is a preferred target region. Similarly, the terms “stop codon region” and “translation termination codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation termination codon. This region is a preferred target region. The open reading frame (ORF) or “coding region,” which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Other preferred target regions include the 5′ untranslated region (5′UTR), known in the art to refer to the portion of an mRNA in the 5′ direction from the translation initiation codon, and thus including nucleotides between the 5′ cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene and the 3′ untranslated region (3′UTR), known in the art to refer to the portion of an mRNA in the 3′ direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3′ end of an mRNA or corresponding nucleotides on the gene. The 5′ cap of an mRNA comprises an N7-methylated guanosine residue joined to the 5′-most residue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA is considered to include the 5′ cap structure itself as well as the first 50 nucleotides adjacent to the cap. The 5′ cap region may also be a preferred target region.

Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as “introns,” which are excised from a pre-mRNA transcript to yield one or more mature mRNA. The remaining (and therefore translated) regions are known as “exons” and are spliced together to form a continuous mRNA sequence. mRNA splice sites, i.e., exon-exon or intron-exon junctions, may also be preferred target regions, and are particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred targets. Targeting particular exons in alternatively spliced mRNAs may also be preferred. It has also been found that introns can also be effective, and therefore preferred, target regions for antisense compounds targeted, for example, to DNA or pre-mRNA.

Once the target site or sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired modulation.

“Hybridization”, in the context of this invention, means hydrogen bonding, also known as Watson-Crick base pairing, between complementary bases, usually on opposite nucleic acid strands or two regions of a nucleic acid strand. Guanine and cytosine are examples of complementary bases which are known to form three hydrogen bonds between them. Adenine and thymine are examples of complementary bases which form two hydrogen bonds between them.

“Specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between the DNA or RNA target and the oligonucleotide.

It is understood that an oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable. An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target interferes with the normal function of the target molecule to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment or, in the case of in vitro assays, under conditions in which the assays are conducted.

Hybridization of antisense oligonucleotides with mRNA interferes with one or more of the normal functions of mRNA. The functions of mRNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in by the RNA. Binding of specific protein(s) to the RNA may also be interfered with by antisense oligonucleotide hybridization to the RNA.

The overall effect of interference with mRNA function is modulation of expression of a Rho family member. In the context of this invention “modulation” means either inhibition or stimulation; i.e., either a decrease or increase in expression. This modulation can be measured in ways which are routine in the art, for example by Northern blot assay of mRNA expression, or reverse transcriptase PCR, as taught in the examples of the instant application or by Western blot or ELISA assay of protein expression, or by an immunoprecipitation assay of protein expression. Effects on cell proliferation or tumor cell growth can also be measured, as taught in the examples of the instant application. Inhibition is presently preferred.

The oligonucleotides of this invention can be used in diagnostics, therapeutics, prophylaxis, and as research reagents and in kits. Since the oligonucleotides of this invention hybridize to nucleic acids encoding a Rho family member, sandwich, colorimetric and other assays can easily be constructed to exploit this fact. Provision of means for detecting hybridization of oligonucleotide with a Rho family member gene or mRNA can routinely be accomplished. Such provision may include enzyme conjugation, radiolabelling or any other suitable detection systems. Kits for detecting the presence or absence of a Rho family member may also be prepared.

The present invention is also suitable for diagnosing abnormal proliferative states in tissue or other samples from patients suspected of having a hyperproliferative disease such as cancer. The ability of the oligonucleotides of the present invention to inhibit cell proliferation may be employed to diagnose such states. A number of assays may be formulated employing the present invention, which assays will commonly comprise contacting a tissue sample with an oligonucleotide of the invention under conditions selected to permit detection and, usually, quantitation of such inhibition. In the context of this invention, to “contact” tissues or cells with an oligonucleotide or oligonucleotides means to add the oligonucleotide(s), usually in a liquid carrier, to a cell suspension or tissue sample, either in vitro or ex vivo, or to administer the oligonucleotide(s) to cells or tissues within an animal. Similarly, the present invention can be used to distinguish a Rho family member-associated tumor from tumors having other etiologies, or those associated with one rho family member from another, in order that an efficacious treatment regimen can be designed.

The oligonucleotides of this invention may also be used for research purposes. Thus, the specific hybridization exhibited by the oligonucleotides may be used for assays, purifications, cellular product preparations and in other methodologies which may be appreciated by persons of ordinary skill in the art.

In the context of this invention, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid or deoxyribonucleic acid. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent intersugar (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced binding to target and increased stability in the presence of nucleases.

The antisense compounds in accordance with this invention preferably comprise from about 5 to about 50 nucleobases. Particularly preferred are antisense oligonucleotides comprising from about 8 to about 30 nucleobases (i.e. from about 8 to about 30 linked nucleosides). As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn the respective ends of this linear polymeric structure can be further joined to form a circular structure, however, open linear structures are generally preferred. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′ . Various salts, mixed salts and free acid forms are also included.

Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361 and 5,625,050.

Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.

Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439.

In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262. Further teaching of PNA compounds can be found in Nielsen et al. (Science, 1991, 254, 1497-1500).

Most preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH2 13 NH—O—CH2—, —CH2—N(CH3)—O—CH2— [known as a methylene (methylimino) or MMI backbone], —CH2—N(CH3)—CH2—, —CH2—N(CH3)—N(CH3)—CH2— and —O—N(CH3)—CH2—CH2— [wherein the native phosphodiester backbone is represented as —O—P—O—CH2—] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2′ position: OH; F; O—, S—, or N-alkyl, O-alkyl-O-alkyl, O—, S—, or N-alkenyl, or O—, S— or N-alkynyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Particularly preferred are O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)2ON(CH3)2, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2′ position: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, poly-alkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2′-DMAOE, as described in U.S. application Ser. No. 09/016,520, filed on Jan. 30, 1998, which is commonly owned with the instant application and the contents of which are herein incorporated by reference.

Other preferred modifications include 2′-methoxy (2′-O—CH3), 2′-aminopropoxy (2′-OCH2CH2CH2NH2) and 2′-fluoro (2′-F). Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugars structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,0531 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920.

Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C or m5c), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia Of Polymer Science And Engineering 1990, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, those disclosed by Englisch et al. (Angewandte Chemie, International Edition 1991, 30, 613-722), and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications 1993, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications 1993, CRC Press, Boca Raton, pages 276-278) and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.: 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; and 5,681,941.

Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett. 1994, 4, 1053-1059), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci. 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let. 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res. 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J. 1991, 10, 1111-1118; Kabanov et al., FEBS Lett. 1990, 259, 327-330; Svinarchuk et al., Biochimie 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett. 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res. 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett. 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther. 1996, 277, 923-937).

Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.

The present invention also includes oligonucleotides which are chimeric oligonucleotides. “Chimeric” oligonucleotides or “chimeras,” in the context of this invention, are oligonucleotides which contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of antisense inhibition of gene expression. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art. This RNAse H-mediated cleavage of the RNA target is distinct from the use of ribozymes to cleave nucleic acids. Ribozymes are not comprehended by the present invention.

Examples of chimeric oligonucleotides include but are not limited to “gapmers,” in which three distinct regions are present, normally with a central region flanked by two regions which are chemically equivalent to each other but distinct from the gap. A preferred example of a gapmer is an oligonucleotide in which a central portion (the “gap”) of the oligonucleotide serves as a substrate for RNase H and is preferably composed of 2′-deoxynucleotides, while the flanking portions (the 5′ and 3′ “wings”) are modified to have greater affinity for the target RNA molecule but are unable to support nuclease activity (e.g., 2′-fluoro- or 2′-O-methoxyethyl-substituted). other chimeras include “wingmers,” also known in the art as “hemimers,” that is, oligonucleotides with two distinct regions. In a preferred example of a wingmer, the 5′ portion of the oligonucleotide serves as a substrate for RNase H and is preferably composed of 2′-deoxynucleotides, whereas the 3′ portion is modified in such a fashion so as to have greater affinity for the target RNA molecule but is unable to support nuclease activity (e.g., 2′-fluoro- or 2′-O-methoxyethyl- substituted), or vice-versa. In one embodiment, the oligonucleotides of the present invention contain a 2′-O-methoxyethyl (2′-O—CH2CH2OCH3) modification on the sugar moiety of at least one nucleotide. This modification has been shown to increase both affinity of the oligonucleotide for its target and nuclease resistance of the oligonucleotide. According to the invention, one, a plurality, or all of the nucleotide subunits of the oligonucleotides of the invention may bear a 2′-O-methoxyethyl (—O—CH2CH2OCH) modification. oligonucleotides comprising a plurality of nucleotide subunits having a 2′-O-methoxyethyl modification can have such a modification on any of the nucleotide subunits within the oligonucleotide, and may be chimeric oligonucleotides. Aside from or in addition to 2′-O-methoxyethyl modifications, oligonucleotides containing other modifications which enhance antisense efficacy, potency or target affinity are also preferred. Chimeric oligonucleotides comprising one or more such modifications are presently preferred.

The oligonucleotides used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including Applied Biosystems. Any other means for such synthesis may also be employed; the actual synthesis of the oligonucleotides is well within the talents of the routineer. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and 2′-alkoxy or 2′-alkoxyalkoxy derivatives, including 2′-O-methoxyethyl oligonucleotides (Martin, P., Helv. Chim. Acta 1995, 78, 486-504). It is also well known to use similar techniques and commercially available modified amidites and controlled-pore glass (CPG) products such as biotin, fluorescein, acridine or psoralen-modified amidites and/or CPG (available from Glen Research, Sterling, Va.) to synthesize fluorescently labeled, biotinylated or other conjugated oligonucleotides.

The antisense compounds of the present invention include bioequivalent compounds, including pharmaceutically acceptable salts and prodrugs. This is intended to encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of the nucleic acids of the invention and prodrugs of such nucleic acids. A pharmaceutically acceptable salts@ are physiologically and pharmaceutically acceptable salts of the nucleic acids of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto (see, for example, Berge et al., “Pharmaceutical Salts,” J. of Pharma Sci. 1977, 66, 1-19).

For oligonucleotides, examples of pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine.

The oligonucleotides of the invention may additionally or alternatively be prepared to be delivered in a Aprodrug@ form. The term Aprodrug® indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligonucleotides of the invention are prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993.

For therapeutic or prophylactic treatment, oligonucleotides are administered in accordance with this invention. Oligonucleotide compounds of the invention may be formulated in a pharmaceutical composition, which may include pharmaceutically acceptable carriers, thickeners, diluents, buffers, preservatives, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients and the like in addition to the oligonucleotide. Such compositions and formulations are comprehended by the present invention.

Pharmaceutical compositions comprising the oligonucleotides of the present invention may include penetration enhancers in order to enhance the alimentary delivery of the oligonucleotides. Penetration enhancers may be classified as belonging to one of five broad categories, i.e., fatty acids, bile salts, chelating agents, surfactants and non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems 1991, 8, 91-192; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems 1990, 7, 1-33). One or more penetration enhancers from one or more of these broad categories may be included.

Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, recinleate, monoolein (a.k.a. 1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, mono- and di-glycerides and physiologically acceptable salts thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems 1990, 7, 1; El-Hariri et al., J. Pharm. Pharmacol. 1992 44, 651-654).

The physiological roles of bile include the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 In: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al., eds., McGraw-Hill, New York, N.Y., 1996, pages 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus, the term “bile salt” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives.

Complex formulations comprising one or more penetration enhancers may be used. For example, bile salts may be used in combination with fatty acids to make complex formulations.

Chelating agents include, but are not limited to, disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines) [Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems 1990, 7, 1-33; Buur et al., J. Control Rel. 1990, 14, 43-51). Chelating agents have the added advantage of also serving as DNase inhibitors.

Surfactants include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems 1991, page 92); and perfluorochemical emulsions, such as FC-43 (Takahashi et al., J. Pharm. Phamacol. 1988, 40, 252-257).

Non-surfactants include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol. 1987, 39, 621-626).

As used herein, “carrier compound” refers to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor.

In contrast to a carrier compound, a “pharmaceutically acceptable carrier” (excipient) is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The pharmaceutically acceptable carrier may be liquid or solid and is selected with the planned manner of administration in mind so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutically acceptable carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrates (e.g., starch, sodium starch glycolate, etc.); or wetting agents (e.g., sodium lauryl sulphate, etc.). Sustained release oral delivery systems and/or enteric coatings for orally administered dosage forms are described in U.S. Pat. Nos. 4,704,295; 4,556,552; 4,309,406; and 4,309,404.

The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional compatible pharmaceutically-active materials such as, e.g., antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the composition of present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the invention.

Regardless of the method by which the oligonucleotides of the invention are introduced into a patient, colloidal dispersion systems may be used as delivery vehicles to enhance the in vivo stability of the oligonucleotides and/or to target the oligonucleotides to a particular organ, tissue or cell type. Colloidal dispersion systems include, but are not limited to, macromolecule complexes, nanocapsules, microspheres, beads and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, liposomes and lipid:oligonucleotide complexes of uncharacterized structure. A preferred colloidal dispersion system is a plurality of liposomes. Liposomes are microscopic spheres having an aqueous core surrounded by one or more outer layers made up of lipids arranged in a bilayer configuration (see, generally, Chonn et al., Current Op. Biotech. 1995, 6, 698-708).

The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, epidermal, and transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, pulmonary administration, e.g., by inhalation or insufflation, or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2′-O-methoxyethyl modification are believed to be particularly useful for oral administration.

Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.

Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.

Compositions for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. In some cases it may be more effective to treat a patient with an oligonucleotide of the invention in conjunction with other traditional therapeutic modalities in order to increase the efficacy of a treatment regimen. In the context of the invention, the term “treatment regimen” is meant to encompass therapeutic, palliative and prophylactic modalities. For example, a patient may be treated with conventional chemotherapeutic agents, particularly those used for tumor and cancer treatment. Examples of such chemotherapeutic agents include but are not limited to daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine,taxol, vincristine, vinblastine, etoposide, trimetrexate, teniposide, cisplatin and diethylstilbestrol (DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. Preferred are chemotherapeutic agents which are direct or indirect inhibitors of a Rho family member. These include MTX, Tomudex and fluorinated pyrimidines such as 5-FU and 5-FUdR. When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide).

The formulation of therapeutic compositions and their subsequent administration is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC50s found to be effective in vitro and in in vivo animal models. In general, dosage is from 0.01 μg to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 μg to 100 g per kg of body weight, once or more daily, to once every 20 years.

Thus, in the context of this invention, by “therapeutically effective amount” is meant the amount of the compound which is required to have a therapeutic effect on the treated individual. This amount, which will be apparent to the skilled artisan, will depend upon the age and weight of the individual, the type of disease to be treated, perhaps even the gender of the individual, and other factors which are routinely taken into consideration when designing a drug treatment. A therapeutic effect is assessed in the individual by measuring the effect of the compound on the disease state in the animal. For example, if the disease to be treated is cancer, therapeutic effects are assessed by measuring the rate of growth or the size of the tumor, or by measuring the production of compounds such as cytokines, production of which is an indication of the progress or regression of the tumor.

The following examples illustrate the present invention and are not intended to limit the same.

EXAMPLES Example 1 Synthesis of Oligonucleotides

Unmodified oligodeoxynucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 380B) using standard phosphoramidite chemistry with oxidation by iodine. β-cyanoethyldiisopropyl-phosphoramidites are purchased from Applied Biosystems (Foster City, Calif.). For phosphorothioate oligonucleotides, the standard oxidation bottle was replaced by a 0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwise thiation of the phosphite linkages. The thiation cycle wait step was increased to 68 seconds and was followed by the capping step.

2′-methoxy oligonucleotides were synthesized using 2′-methoxy β-cyanoethyldiisopropyl-phosphoramidites (Chemgenes, Needham, Mass.) and the standard cycle for unmodified oligonucleotides, except the wait step after pulse delivery of tetrazole and base was increased to 360 seconds. Other 2′-alkoxy oligonucleotides were synthesized by a modification of this method, using appropriate 2′-modified amidites such as those available from Glen Research, Inc., Sterling, Va.

2′-fluoro oligonucleotides were synthesized as described in Kawasaki et al. (J. Med. Chem. 1993, 36, 831-841). Briefly, the protected nucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesized utilizing commercially available 9-β-D-arabinofuranosyladenine as starting material and by modifying literature procedures whereby the 2′-α-fluoro atom is introduced by a SN2-displacement of a 2′-β-O-trifyl group. Thus N6-benzoyl-9-62 -D-arabinofuranosyladenine was selectively protected in moderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate. Deprotection of the THP and N6-benzoyl groups was accomplished using standard methodologies and standard methods were used to obtain the 5′-dimethoxytrityl- (DMT) and 5′-DMT-3′-phosphoramidite intermediates.

The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplished using tetraisopropyldisiloxanyl (TPDS) protected 9-β-D-arabinofuranosylguanine as starting material, and conversion to the intermediate diisobutyryl-arabinofuranosylguanosine. Deprotection of the TPDS group was followed by protection of the hydroxyl group with THP to give diisobutyryl di-THP protected arabinofuranosylguanine. Selective O-deacylation and triflation was followed by treatment of the crude product with fluoride, then deprotection of the THP groups. Standard methodologies were used to obtain the 5′-DMT- and 5′-DMT-3′-phosphoramidites.

Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by the modification of a known procedure in which 2,2′-anhydro-1-β-D-arabinofuranosyluracil was treated with 70% hydrogen fluoride-pyridine. Standard procedures were used to obtain the 5′-DMT and 5′-DMT-3′ phosphoramidites.

2′-deoxy-2′-fluorocytidine was synthesized via amination of 2′-deoxy-2′-fluorouridine, followed by selective protection to give N4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used to obtain the 5′-DMT and 5′-DMT-3′ phosphoramidites.

2′-(2-methoxyethyl)-modified amidites are synthesized according to Martin, P. (Helv. Chim. Acta 1995, 78, 486-506). For ease of synthesis, the last nucleotide was a deoxynucleotide. 2′-O—CH2CH2OCH3-cytosines may be 5-methyl cytosines.

Synthesis of 5-Methyl Cytosine Monomers

2.2′-Anhydro[1-(β-D-arabinofuranosyl)-5-methyluridinel

5-Methyluridine (ribosylthymine, commercially available through Yamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenyl-carbonate (90.0 g, 0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). The mixture was heated to reflux, with stirring, allowing the evolved carbon dioxide gas to be released in a controlled manner. After 1 hour, the slightly darkened solution was concentrated under reduced pressure. The resulting syrup was poured into diethylether (2.5 L), with stirring. The product formed a gum. The ether was decanted and the residue was dissolved in a minimum amount of methanol (ca. 400 mL). The solution was poured into fresh ether (2.5 L) to yield a stiff gum. The ether was decanted and the gum was dried in a vacuum oven (60° C. at 1 mm Hg for 24 h) to give a solid which was crushed to a light tan powder (57 g, 85% crude yield). The material was used as is for further reactions.

2′-O-Methoxyethyl-5-methyluridine

2,2′-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol (1.2 L) were added to a 2 L stainless steel pressure vessel and placed in a pre-heated oil bath at 160° C. After heating for 48 hours at 155-160° C., the vessel was opened and the solution evaporated to dryness and triturated with MeOH (200 mL). The residue was suspended in hot acetone (1 L). The insoluble salts were filtered, washed with acetone (150 mL) and the filtrate evaporated. The residue (280 g) was dissolved in CH3CN (600 mL) and evaporated. A silica gel column (3 kg) was packed in CH2Cl2/acetone/MeOH (20:5:3) containing 0.5% Et3NH. The residue was dissolved in CH2Cl2 (250 mL) and adsorbed onto silica (150 g) prior to loading onto the column. The product was eluted with the packing solvent to give 160 g (63%) of product.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporated with pyridine (250 mL) and the dried residue dissolved in pyridine (1.3 L). A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and the mixture stirred at room temperature for one hour. A second aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and the reaction stirred for an additional one hour. Methanol (170 mL) was then added to stop the reaction. HPLC showed the presence of approximately 70% product. The solvent was evaporated and triturated with CH3CN (200 mL). The residue was dissolved in CHCl3 (1.5 L) and extracted with 2×500 mL of saturated NaHCO3 and 2×500 mL of saturated NaCl. The organic phase was dried over Na2SO4, filtered and evaporated. 275 g of residue was obtained. The residue was purified on a 3.5 kg silica gel column, packed and eluted with EtOAc/-Hexane/Acetone (5:5:1) containing 0.5% Et3NH. The pure fractions were evaporated to give 164 g of product. Approximately 20 g additional was obtained from the impure fractions to give a total yield of 183 g (57%).

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-uridine

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and 188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) were combined and stirred at room temperature for 24 hours. The reaction was monitored by tlc by first quenching the tlc sample with the addition of MeOH. Upon completion of the reaction, as judged by tlc, MeOH (50 mL) was added and the mixture evaporated at 35° C. The residue was dissolved in CHCl3 (800 mL) and extracted with 2×200 mL of saturated sodium bicarbonate and 2×200 mL of saturated NaCl. The water layers were back extracted with 200 mL of CHCl3. The combined organics were dried with sodium sulfate and evaporated to give 122 g of residue (approx. 90% product). The residue was purified on a 3.5 kg silica gel column and eluted using EtOAc/Hexane(4:1). Pure product fractions were evaporated to yield 96 g (84%).

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine

A first solution was prepared by dissolving 3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96 g, 0.144 M) in CH3CN (700 mL) and set aside. Triethylamine (189 mL, 1.44 M) was added to a solution of triazole (90 g, 1.3 M) in CH3CN (1 L), cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POCl3 was added dropwise, over a 30 minute period, to the stirred solution maintained at 0-10° C., and the resulting mixture stirred for an additional 2 hours. The first solution was added dropwise, over a 45 minute period, to the later solution. The resulting reaction mixture was stored overnight in a cold room. Salts were filtered from the reaction mixture and the solution was evaporated. The residue was dissolved in EtOAc (1 L) and the insoluble solids were removed by filtration. The filtrate was washed with 1×300 mL of NaHCO3 and 2×300 mL of saturated NaCl, dried over sodium sulfate and evaporated. The residue was triturated with EtOAc to give the title compound.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

A solution of 3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine (103 g, 0.141 M) in dioxane (500 mL) and NH4OH (30 mL) was stirred at room temperature for 2 hours. The dioxane solution was evaporated and the residue azeotroped with MeOH (2×200 mL). The residue was dissolved in MeOH (300 mL) and transferred to a 2 liter stainless steel pressure vessel. MeOH (400 mL) saturated with NH3 gas was added and the vessel heated to 100° C. for 2 hours (tlc showed complete conversion). The vessel contents were evaporated to dryness and the residue was dissolved in EtOAc (500 mL) and washed once with saturated NaCl (200 mL). The organics were dried over sodium sulfate and the solvent was evaporated to give 85 g (95%) of the title compound.

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-cytidine

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M) was added with stirring. After stirring for 3 hours, tlc showed the reaction to be approximately 95% complete. The solvent was evaporated and the residue azeotroped with MeOH (200 mL). The residue was dissolved in CHCl3 (700 mL) and extracted with saturated NaHCO, (2×300 mL) and saturated NaCl (2×300 mL), dried over MgSO, and evaporated to give a residue (96 g). The residue was chromatographed on a 1.5 kg silica column using EtOAc/Hexane (1:1) containing 0.5% Et3NH as the eluting solvent. The pure product fractions were evaporated to give 90 g (90%) of the title compound.

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74 g, 0.10 M) was dissolved in CH2Cl2 (1 L) . Tetrazole diisopropylamine (7.1 g) and 2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M) were added with stirring, under a nitrogen atmosphere. The resulting mixture was stirred for 20 hours at room temperature (tlc showed the reaction to be 95% complete) The reaction mixture was extracted with saturated NaHCO3 (1×300 mL) and saturated NaCl (3×300 mL). The aqueous washes were back-extracted with CH2Cl2 (300 mL) , and the extracts were combined, dried over MgSO4 and concentrated. The residue obtained was chromatographed on a 1.5 kg silica column using EtOAc\Hexane (3:1) as the eluting solvent. The pure fractions were combined to give 90.6 g (87%) of the title compound.

5-methyl-2′-deoxycytidine (5-me-C) containing oligonucleotides were synthesized according to published methods (Sanghvi et al., Nucl. Acids Res. 1993, 21, 3197-3203) using commercially available phosphoramidites (Glen Research, Sterling Va. or ChemGenes, Needham Mass.).

Oligonucleotides having methylene(methylimino) (MMI) backbones are synthesized according to U.S. Pat. No. 5,378,825, which is coassigned to the assignee of the present invention and is incorporated herein in its entirety. For ease of synthesis, various nucleoside dimers containing MMI linkages were synthesized and incorporated into oligonucleotides. other nitrogen-containing backbones are synthesized according to WO 92/20823 which is also coassigned to the assignee of the present invention and incorporated herein in its entirety.

Oligonucleotides having amide backbones are synthesized according to De Mesmaeker et al. (Acc. Chem. Res. 1995, 28, 366-374). The amide moiety is readily accessible by simple and well-known synthetic methods and is compatible with the conditions required for solid phase synthesis of oligonucleotides.

Oligonucleotides with morpholino backbones are synthesized according to U.S. Pat. No. 5,034,506 (Summerton and Weller).

Peptide-nucleic acid (PNA) oligomers are synthesized according to P. E. Nielsen et al. (Science 1991, 254, 1497-1500).

After cleavage from the controlled pore glass column (Applied Biosystems) and deblocking in concentrated ammonium hydroxide at 55° C. for 18 hours, the oligonucleotides are purified by precipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol. Synthesized oligonucleotides were analyzed by polyacrylamide gel electrophoresis on denaturing gels and judged to be at least 85% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in synthesis were periodically checked by 31P nuclear magnetic resonance spectroscopy, and for some studies oligonucleotides were purified by HPLC, as described by Chiang et al. (J. Biol. Chem. 1991, 266, 18162). Results obtained with HPLC-purified material were similar to those obtained with non-HPLC purified material.

Example 2 Human RhoA Oligonucleotide Sequences

Antisense oligonucleotides were designed to target human RhoA. Target sequence data are from the RhoA cDNA sequence published by Yeramian, P., et al. (Nucleic Acids Res. 1987, 15, 1869); Genbank accession number X05026, provided herein as SEQ ID NO: 1. Oligonucleotides were synthesized primarily with phosphorothioate linkages. Oligonucleotide sequences are shown in Table 1.

A549 cells, human lung carcinoma cells (obtained from American Type Culture Collection) were cultured in Dulbecco's modified Eagle's medium (DMEM) low glucose, 10% fetal calf serum, and penicillin (50 units/ml)/streptomycin (50 mg/ml). Cells were passaged at 90-956 confluency. All culture reagents were obtained from Life Technologies (GIBCO BRL, Rockville, Md.).

A549 cells were plated at a starting cell number of approximately 2×105 cells per well. After twenty-four hours, at 80-90% confluency, the cells were washed twice with Opti-Mem (GIBCO BRL) and the oligonucleotide formulated in LIPOFECTIN (GIBCO BRL), a 1:1 (w/w) liposome formulation of the cationic lipid N-(1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA), and dioleoyl phosphotidylethanolamine (DOPE) in membrane filtered water, at a constant ratio of 2.5 mg/ml LIPOFECTIN to 100 nM oligonucleotide, in Opti-Mem. For an initial screen, the oligonucleotide concentration was 300 nM. Treatment was for four hours. After treatment, the media was removed and the cells were further incubated in DMEM containing 10% FCS, and penicillin/streptomycin for 24 or 48 hours.

mRNA was isolated using the MICRO-FASTTRACK kit (Invitrogen, Carlsbad, Calif.), separated on a 1% agarose gel, transferred to Hybond-N+ membrane (Amersham, Arlington Heights, Ill.), a positively charged nylon membrane, and probed. A RhoA probe was generated using asymmetric PCR, in the presence of a [32P]-dCTP (Amersham), with the following primers:

Forward: 5′-TGCAAGCACAGCCCTTATG-3′ SEQ ID NO. 2

Reverse: 5′-TGTCAAAGGACCCTGGTG-3′ SEQ ID NO. 3

A glyceraldehyde 3-phosphate dehydrogenase (G3PDH) probe was purchased from Clontech (Palo Alto, Calif.), Catalog Number 9801-1. The probe was labeled by random primer using the Large Fragment of DNA polymerase (Klenow fragment) (GIBCO BRL) as described in Maniatis, T., et al., Molecular Cloning: A Laboratory Manual, 1989. mRNA was quantitated by a PhosphoImager (Molecular Dynamics, Sunnyvale, Calif.).

TABLE 1
Nucleotide Sequences of RhoA Oligonucleotides
TARGET GENE
SEQ NUCLEOTIDE GENE
ISIS NUCLEOTIDE SEQUENCE ID CO- TARGET
NO. (5′ -> 3′) NO: ORDINATES1 REGION
16191 AGTCGCAAACTCGGAGAC 4 0085-0102 5′-UTR
16192 TTGCTCAGGCAACGAATC 5 0142-0159 AUG
16193 CTGAAGACTATGAGCAAGCATG 6 0214-0235 Coding
16194 CTCATCATTCCGAAGATCC 7 0515-0533 Coding
16195 CCAATCCTGTTTGCCATATCTC 8 0592-0613 Coding
16196 CCATCTTTGGTCTTTGCTGAAC 9 0634-0655 Coding
16197 GCAGAGCAGCTCTCGTAGCCA 10 0676-0696 Coding
16198 TCACAAGACAAGGCAACCAG 11 0721-0740 Stop
16199 AGGCCAGTAATCATACACTA 12 0799-0818 3′-UTR
16200 GTTGGCTTCTAAATACTGGT 13 0871-0890 3′-UTR
16201 GGCTGTTAGAGCAGTGTCAA 14 0937-0956 3′-UTR
16202 AGCGCCTGGTGTGTCAGGTG 15 0971-0990 3′-UTR
16203 TAGTTACAGCCTAATTGACA 16 1051-1073 3′-UTR
16913 GGCACCTGTTGGGTGAGCTG 17 16202 control
16914 ACACTCTTGCTTACCGTACCTT 18 16195 control
16915 TGCGGTAAGTGCGGTATCAA 19 16201 control
1All linkages are phosphorothioate linkages.
2Co-ordinates from Genbank Accession No. X05026, locus name “HSRHOB” SEQ ID NO. 1.

Results are shown in Table 2. Oligonucleotides 16193 (SEQ ID NO. 6), 16195 (SEQ ID NO. 8), 16196 (SEQ ID NO. 9), 16197 (SEQ ID NO. 10), 16198 (SEQ ID NO. 11), 16199 (SEQ ID NO. 12), 16200 (SEQ ID NO. 13), 16201 (SEQ ID NO. 14), and 16202 (SEQ ID NO. 15) gave better than 50% inhibition of RhoA expression. Oligonucleotides 16195 (SEQ ID NO. 8), 16197 (SEQ ID NO. 10), 16199 (SEQ ID NO. 12), 16201 (SEQ ID NO. 14), and 16202 (SEQ ID NO. 15) gave better than 75% inhibition of RhoA expression.

TABLE 2
Activities of Phosphorothioate Oligonucleotides Targeted to
Human RhoA
SEQ GENE
ISIS ID TARGET % mRNA % mRNA
No: NO: REGION EXPRESSION INHIBITION
LIPOFECTIN 100.0%   0.0%
only
16191 4 5′-UTR 66.4% 33.6%
16192 5 AUG 68.0% 32.0%
16193 6 Coding 31.9% 68.1%
16194 7 Coding 79.9% 20.1%
16195 8 Coding  3.9% 96.1%
16196 9 Coding 31.4% 68.6%
16197 10 Coding 19.2% 81.8%
16198 11 Stop 46.4% 53.6%
16199 12 3′-UTR 22.9% 77.1%
16200 13 3′-UTR 36.9% 63.1%
16201 14 3′-UTR 22.0% 78.0%
16202 15 3′-UTR 14.4% 85.6%
16203 16 3′-UTR 88.0% 12.0%

Example 3 Dose Response and Specificity of Antisense Oligonucleotide Effects on Human RhoA mRNA Levels in A549 Cells

Three of the most active oligonucleotides from the initial screen were chosen for dose response assays. These include oligonucleotides 16195 (SEQ ID NO. 8), 16201 (SEQ ID NO. 14), and 16202 (SEQ ID NO. 15). A549 cells were grown, treated and processed as described in Example 2. LIPOFECTIN was added at a ratio of 2.5 mg/ml per 100 nM of oligonucleotide. The control included LIPOFECTIN at a concentration of 7.5 mg/ml. Results are shown in Table 3. Each oligonucleotide showed a dose response effect with in maximal inhibition greater than 90%.

The specificity of these oligonucleotides was investigated using scrambled controls, i.e. oligonucleotides with the same base composition and a scrambled sequence. Oligonucleotide 16915 (SEQ ID NO. 19) is a scrambled control for 16201 (SEQ ID NO. 14) and oligonucleotide 16913 (SEQ ID NO. 17) is a scrambled control for 16202 (SEQ ID NO. 15). Both antisense oligonucleotides showed a dose dependent effect on mRNA expression, while scrambled controls showed much less inhibition which was only seen at higher does.

TABLE 3
Dose Response of A549 Cells to RhoA
Antisense Oligonucleotides (ASOs)
SEQ ID ASO Gene % mRNA % mRNA
ISIS # NO: Target Dose Expression Inhibition
control LIPOFECTIN 100.0%   0.0%
only
16195 8 Coding  75 nM 72.7% 27.3%
16195 8 150 nM 35.0% 65.0%
16195 8 300 nM 20.3% 79.7%
16201 14 3′-UTR  75 nM 79.1% 20.9%
16201 14 150 nM 35.7% 64.3%
16201 14 300 nM  9.5% 90.5%
16202 15 3′-UTR  75 nM 68.7% 31.3%
16202 15 150 nM 28.8% 71.2%
16202 15 300 nM  6.1% 93.7%

TABLE 4
Specificity of RhoA Antisense Oligonucleotides (ASOs) in
A549 Cells
SEQ ID ASO Gene % mRNA % mRNA
ISIS # NO: Target Dose Expression Inhibition
control LIPOFECTIN  100%   0%
only
16201 14 3′-UTR  75 nM 64.4% 35.6%
16201 14 150 nM 35.3% 64.7%
16201 14 300 nM  5.7% 94.3%
16915 19 control  75 nM 89.9% 10.1%
16915 19 150 nM 98.3%  1.7%
16915 19 300 nM 84.8% 15.2%
16202 15 3′-UTR  75 nM 39.9% 60.1%
16202 15 150 nM 20.2% 79.8%
16202 15 300 nM 10.8% 89.2%
16913 17 control  75 nM 97.6%  2.4%
16913 17 150 nM 89.8% 10.2%
16913 17 300 nM 55.6% 44.4%

Example 4

Design and Testing of Chimeric (Deoxy Gapped) 2′-O-methoxyethyl RhoA Antisense Oligonucleotides on RhoA Levels in A549 Cells

Oligonucleotides having SEQ ID NO: 14 were synthesized as a uniformly phosphorothioate or mixed phosphorothioate/phosphodiester chimeric oligonucleotides having variable regions of 2′-O-methoxyethyl (2′-MOE) nucleotides and deoxynucleotides. All 2′-MOE cytosines were 5-methyl-cytosines. Additionally, some oligonucleotides were synthesized with deoxycytosines as 5-methyl-cytosines. Additional oligonucleotides were synthesized, with similar chemistries, as scrambled controls.

TABLE 5
Nucleotide Sequences of 16201 Analogues
SEQ TARGET GENE GENE
ISIS NUCLEOTIDE SEQUENCE ID NUCLEOTIDE TARGET
NO. (5′ -> 3′)1 NO: CO-ORDINATES2 REGION
17130 GsGsCsTsGsTsTsAsGsAsGsCsAsGsTsGsTsCsAsA 14 0937-0956 3′-UTR
17131 GsGsCsTsGsTsTsAsGsAsGsCsAsGsTsGsTsCsAsA 14 0937-0956 3′-UTR
17132 GsGsCsTsGsTsTsAsGsAsGsCsAsGsTsGsTsCsAsA 14 0937-0956 3′-UTR
17133 GsGsCsTsGsTsTsAsGsAsGsCsAsGsTsGsTsCsAsA 14 0937-0956 3′-UTR
17134 GsGsCsTsGsTsTsAsGsAsGsCsAsGsTsGsTsCsAsA 14 0937-0956 3′-UTR
17818 GoGoCsTsGsTsTsAsGsAsGsCsAoGoToGoToCoAoA 14 0937-0956 all 5-meC
17819 ToGoCsGsGsTsAsAsGsTsGsCsGoGoToAoToCoAoA 19 16201 control all 5-meC
18550 TsGsCsGsGsTsAsAsGsTsGsCsGsGsTsAsTsCsAsA 19 16201 control
20459 GsGsCsTsGsTsTsAsGsAsGsCsAsGsTsGsTsCsAsA 14 0937-0956 all 5-meC
21919 GsTsCsGsTsTsAsGsTsCsGsAsAsAsTsGsAsGsGsC 20 16201 control
21920 AsGsCsTsTsGsTsGsAsAsCsGsAsGsTsGsTsCsGsA 21 16201 control
21921 TsGsCsAsGsTsTsGsGsCsAsGsAsGsTsCsTsGsAsA 22 16201 control
1 Emboldened residues are 2′-methoxyethoxy residues (others are 2′-deoxy). A11 2′-methoxyethoxy cytidines are 5-methyl-cytidines; where indicated “all 5-meC”, 2′-deoxycytidines are also 5-methyl-cytidines; “s” linkages are phosphorothioate linkages, “o” linkages are phosphodiester linkages.
2Co-ordinates from Genbank Accession No. X05026, locus name “HSRHOB” SEQ ID NO. 1.

Dose response experiments were performed using chimeric oligonucleotides as discussed in Example 3. Results are shown in Table 6. The introduction of 2′-MOE nucleotides into the sequence improved the maximum inhibition from 60%, with a phosphorothioate oligodeoxynucleotide, to greater than 75%. The exception was the fully modified 2-MOE oligonucleotide which was less effective than the oligodeoxynucleotide.

TABLE 6
Dose Response of A549 Cells to RhoA
Antisense Gapmer Oligonucleotides (ASOs)
SEQ ID ASO Gene % mRNA % mRNA
ISIS # NO: Target Dose Expression Inhibition
control LIPOFECTIN  100%   0%
only
16201 14 3′-UTR  75 nM 119.5% 
16201 14 150 nM 54.5% 45.5%
16201 14 300 nM 39.5% 60.5%
17130 14 3′-UTR  75 nM 56.2% 43.8%
17130 14 150 nM 31.5% 68.5%
17130 14 300 nM 14.1% 85.9%
17131 14 3′-UTR  75 nM 55.5% 44.5%
17131 14 150 nM 35.4% 64.6%
17131 14 300 nM 24.7% 75.3%
17132 14 3′-UTR  75 nM 71.3% 28.7%
17132 14 150 nM 31.3% 68.7%
17132 14 300 nM 13.1% 86.9%
17133 14 3′-UTR  75 nM 41.7% 58.3%
17133 14 150 nM 33.8% 66.2%
17133 14 300 nM 14.4% 85.6%
17134 14 3′-UTR  75 nM 76.6% 23.4%
17134 14 150 nM 35.9% 64.1%
17134 14 300 nM 68.5% 31.5%

Example 5

Time Course of Antisense Oligonucleotide Effects on Human RhoA Protein Levels in A549 Cells

Oligonucleotide 17131 was tested by treating for varying times and measuring the effect of the oligo on RhoA protein levels. A549 cells were grown and treated with oligonucleotide (300 nM) as described in Example 2. Cells were harvested at 24, 48 and 72 hours after treatment. RhoA protein levels were measured by Western blotting. After oligonucleotide treatment, cells were washed twice in phosphate-buffered saline (PBS) and lysed in 25 mM Tris-HCl pH 7.5, 1% Triton X-100, 0.2% SDS, 0.5% sodium deoxycholate, 450 mM NaCl, and 10 mg/ml aprotinin and leupeptin. After 15 minutes on ice, the samples were centrifuged at maximum speed in a microfuge. Protein concentration was determined by Bradford reagent (Bio-Rad Laboratories, Hercules, Calif.). Fifty mg of protein was separated by SDS-PAGE (15%). Following electrophoresis, proteins were transferred to IMMOBILON-P membranes (Millipore, Bedford, Mass.) The membrane was blocked in 5% fish gelatin (Sigma Chemicals, St. Louis, Mo.) and RhoA specific antibodies were used to visualize the proteins. After incubation with the appropriate secondary antibody, proteins were visualized using either LUMIGLO Reagent (New England Biolabs, Beverly, Mass.) or ECL PLUS (Amersham Pharmacia Biotech, Piscataway, N.J.). Inhibition of RhoA protein was observable after 24 hours. After 48 hours, RhoA protein concentration was reduced by 80% using 17131 (SEQ ID NO. 14). Minimal inhibition was seen with 17163 (SEQ ID NO. 190), an oligonucleotide targeted to Rac1. Results are shown in Table 7.

TABLE 7
Time course of RhoA Antisense Oligonucleotides (ASOs) in
A549 Cells
Time
SEQ ID ASO Gene after % protein % protein
ISIS # NO: Target treatmt Expression Inhibition
control LIPOFECTIN  100%   0%
only
17131 14 3′-UTR 24 hr 46.2% 53.8%
17131 14 48 hr 16.0% 84.0%
17131 14 72 hr 12.4% 87.6%
17163 190 Rac1 control 24 hr 104.1% 
17163 190 48 hr 82.3% 17.7%
17163 190 72 hr 95.2%  4.8%

Example 6

Dose Response of Antisense Oligonucleotide Effects on Human RhoA Protein Levels in A549 Cells

Oligonucleotide 17131 was tested for a dose response by using varying concentrations of oligonucleotide and measuring the effect of the oligonucleotide on RhoA protein levels. A549 cells were grown and treated with oligonucleotide (concentrations indicated in Table 8) as described in Example 2. Western blotting was performed to measure protein levels as described in Example 5. A dose response effect is seen with 17131 (SEQ ID NO. 14), whereas the scrambled control 18550 (SEQ ID NO. 19) had no effect on RhoA protein levels.

TABLE 8
Dose response of RhoA antisense oligonucleotide on protein
levels in A549 cells
SEQ ID ASO Gene % protein % protein
ISIS # NO: Target Dose Expression Inhibition
control LIPOFECTIN 100%  0%
only
17131 14 3′-UTR  75 nM 51% 49%
17131 14 150 nM 23% 77%
17131 14 300 nM 20% 80%
18550 19 control  75 nM 101%
18550 19 150 nM 101%
18550 19 300 nM 104%

Example 7

Inhibition of JNK Activation by RhoA Antisense Oligonucleotides in A549 Cells Stimulated with H2O2

Oligonucleotide 17131 (SEQ ID NO. 14) was tested for its ability to inhibit JNK activation by stimulation with H2O2 or Il-1b. A549 cells were grown as described in Example 2. Cells were treated with 150 nM of oligonucleotide for four hours. After treatment, the media was replaced with DMEM, 0.1% FCS, and the cells were left in culture for 48 hours prior to stimulation. Stimulation was with either 30 ng/ml IL-1b or 1 mM H2O2 for 30 minutes. After stimulation, the cells were washed twice in PBS, and lysed in 25 mM Hepes pH 7.7, 0.3 M NaCl, 1.5 mM MgCl2, 0.1% Triton X-100, 20 mM b-glycerophosphate, 0.1 mM sodium orthovanadate (Na3VO4), 0.5 mM PMSF, and 10 mg/ml of aprotinin and leupeptin. After 20 minutes on ice, the lysates were centrifuged at maximum speed in a microfuge for 20 minutes. The protein concentration in the supernatant was determined using Bradford reagent (Bio-Rad Laboratories, Hercules, Calif.). To 150 mg of protein, 25 ml of c-Jun fusion beads (New England Biolabs, Beverly, Mass.) were added and incubated at 4° C. on a rotating wheel overnight. The samples were then washed four times in 20 mM Hepes pH 7.7, 50 mM NaCl, 0.1 mM EDTA, 2.5 mM MgCl2, and 0.05% Triton X-100 (HIBI buffer). The kinase reaction was run for 20 minutes at 30° C. in 20 mM Hepes pH 7.7, 20 mM MgCl2, 20 mM b-glycerophosphate, 20 mM p-nitrophenyl phosphate, 0.1 mM Na3VO4, 2 mM DTT, 20 mM ATP, and 5 mCi of g[32P]-ATP. The reaction was stopped with 500 ml of ice cold HIBI buffer. The beads were pelleted, resuspended in PAGE loading buffer, boiled for 5 minutes, and the products separated on a 12% SDS gel (Novex, La Jolla, Calif.). Bands were quantitated using a PhosphorImager.

Results are shown in Table 9. Oligonucleotide 17131 (SEQ ID NO. 14) showed moderate but specific inhibition of H2O2-induced JNK activation.

TABLE 9
Inhibition of JNK activation by RhoA antisense
oligonucleotides
SEQ
ID ASO Gene % inhibition % inhibition
ISIS # NO: Target Dose Il-1b induced H2O2 induced
control LIPOFECTIN   0%   0%
only
17131 14 3′-UTR 150 nM 37.6%
18550 19 control 150 nM 2.2%  5.8%

Example 8

Synthesis of Additional RhoA Sequences

Additional oligonucleotides were synthesized in 96 well plate format via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a standard 96 well format. Phosphodiester internucleotide linkages were afforded by oxidation with aqueous iodine. Phosphorothioate internucleotide linkages were generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile-. Standard base-protected beta-cyanoethyl-di-isopropyl phosphoramidites were purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.) Non-standard nucleosides are synthesized as per published methods. They are utilized as base protected beta-cyanoethyldiisopropyl phosphoramidites.

Oligonucleotides were cleaved from support and deprotected with concentrated NH4OH at elevated temperature (55-60° C.) for 12-16 hours and the released product then dried in vacuo. The dried product was then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.

A series of oligonucleotides were designed to target different regions of the human RhoA RNA, using published sequences (GenBank accession number X05026, incorporated herein as SEQ ID NO: 1). The oligonucleotides are shown in Table 10. Target sites are indicated by nucleotide numbers, as given in the sequence source reference (Genbank accession no. X05026), to which the oligonucleotide binds.

All compounds in Table 10 are oligodeoxynucleotides with phosphorothioate backbones (internucleoside linkages) throughout. All compounds in Table 11 are chimeric oligonucleotides (“gapmers”) 18 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by four-nucleotide “wings.” The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. Cytidine residues in the 2′-MOE wings are 5-methylcytidines.

TABLE 10
Nucleotide Sequences of Human RhoA
Phosphorothioate Oligodeoxynucleotides
SEQ TARGET GENE GENE
ISIS NUCLEOTIDE SEQUENCE ID NUCLEOTIDE TARGET
NO. (5′ -> 3′) NO: CO-ORDINATES1 REGION
25544 AGAGAACCGACGGAGGAC 23 0030-0047 5′-UTR
25545 GTGGACTAATGAGAGAAC 24 0041-0058 5′-UTR
25546 GACCGTGGACTAATGAGA 25 0045-0062 5′-UTR
25547 AGCTGAAGACCAGACCGT 26 0057-0074 5′-UTR
25548 AGTCGCAAACTCGGAGAC 4 0085-0102 5′-UTR
25549 AATCCGAGTCCAGCCTCT 27 0128-0145 5′-UTR
25550 AACGAATCCGAGTCCAGC 28 0132-0149 5′-UTR
25551 TCAGGCAACGAATCCGAG 29 0138-0155 5′-UTR
25552 CACCAACAATCACCAGTT 30 0178-0195 Coding
25553 AAGACTATGAGCAAGCAT 31 0215-0232 Coding
25554 ATACACCTCTGGGAACTG 32 0243-0260 Coding
25555 ACATAGTTCTCAAACACT 33 0269-0286 Coding
25556 ACTCTACCTGCTTTCCAT 34 0304-0321 Coding
25557 CACAAAGCCAACTCTACC 35 0314-0331 Coding
25558 AACATCGGTATCTGGGTA 36 0378-0395 Coding
25559 TTCTGGGATGTTTTCTAA 37 0432-0449 Coding
25560 GGACAGAAATGCTTGACT 38 0464-0481 Coding
25561 GTGCTCATCATTCCGAAG 39 0519-0536 Coding
25562 CTTGTGTGCTCATCATTC 40 0524-0541 Coding
25563 TAGCTCCCGCCTTGTGTG 41 0534-0551 Coding
25564 CCAATCCTGTTTGCCATA 42 0596-0613 Coding
25565 GTCTTTGCTGAACACTCC 43 0629-0646 Coding
25566 AAAACCTCTCTCACTCCA 44 0653-0670 Coding
25567 AAGACAAGGCAACCAGAT 45 0719-0736 Coding
25568 TTTCACAAGACAAGGCAA 46 0725-0742 Stop

Example 9

Total RNA Isolation

Total mRNA was isolated using an RNEASY 96 kit and buffers purchased from Qiagen Inc. (Valencia Calif.) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 100 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 100 μL of 70% ethanol was then added to each well and the contents mixed by pippeting three times up and down. The samples were then transferred to the RNEASY 96 well plate attached to a QIAVAC manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 15 seconds. 1 mL of Buffer RW1 was added to each well of the RNEASY 96 plate and the vacuum again applied for 15 seconds. 1 mL of Buffer RPE was then added to each well of the RNEASY 96 plate and the vacuum applied for a period of 15 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 10 minutes. The plate was then removed from the QIAVAC manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVAC manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 60 μL water into each well, incubating 1 minute, and then applying the vacuum for 30 seconds. The elution step was repeated with an additional 60 μL water.

Poly(A)+ mRNA may be isolated according to Miura et al., Clin. Chem., 42, 1758 (1996). other methods for poly(A)+ mRNA isolation are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc., (1993). Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 ml cold PBS. 60 ml lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 ml of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 ml of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 ml of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70° C. was added to each well, the plate was incubated on a 90° hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.

Example 10

Real-time Quantitative PCR Analysis of RhoA mRNA Levels

Quantitation of RhoA mRNA levels was determined by real-time quantitative PCR using the ABI PRISM 7700 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR, in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., JOE or FAM, obtained from either Operon Technologies Inc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) is attached to the 5′ end of the probe and a quencher dye (e.g., TAMRA, obtained from either Operon Technologies Inc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) is attached to the 3′ end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3′ quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5′-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular (six-second) intervals by laser optics built into the ABI PRISM 7700 Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.

PCR reagents were obtained from PE-Applied Biosystems, Foster City, Calif. RT-PCR reactions were carried out by adding 25 μL PCR cocktail (1x TAQMAN buffer A, 5.5 mM MgCl2, 300 μM each of dATP, dCTP and dGTP, 600 μM of dUTP, 100 nM each of forward primer, reverse primer, and probe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD, and 12.5 Units MuLV reverse transcriptase) to 96 well plates containing 25 μL poly(A) mRNA solution. The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the AMPLITAQ GOLD, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension). RhoA probes and primers were designed to hybridize to the human RhoA sequence, using published sequence information (GenBank accession number X05026, incorporated herein as SEQ ID NO:1).

For RhoA the PCR primers were:

forward primer: GGCTGGACTCGGATTCGTT (SEQ ID NO: 62)

reverse primer: CCATCACCAACAATCACCAGTT (SEQ ID NO: 63) and the

PCR probe was: FAM-CCTGAGCAATGGCTGCCATCCG-TAMRA

(SEQ ID NO: 64) where FAM (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.

For GAPDH the PCR primers were:

forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 65)

reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 66)and the

PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC- TAMRA 3′ (SEQ ID NO: 67) where JOE (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.

Example 11

Antisense Inhibition of RhoA Expression-phosphorothioate Oligodeoxynucleotides

In accordance with the present invention, a series of oligonucleotides were designed to target different regions of the human RhoA RNA, using published sequences (GenBank accession number X05026, incorporated herein as SEQ ID NO: 1). The oligonucleotides are shown in Table 10. Target sites are indicated by nucleotide numbers, as given in the sequence source reference (Genbank accession no. X05026), to which the oligonucleotide binds. All compounds in Table 10 are oligodeoxynucleotides with phosphorothioate backbones (internucleoside linkages) throughout. The compounds were analyzed for effect on RhoA mRNA levels by quantitative real-time PCR as described in other examples herein. Data are shown in Table 11 and are averages from three experiments. If present, “N.D.” indicates “no data”.

TABLE 11
Inhibition of RhoA mRNA levels by phosphorothioate
oligodeoxynucleotides
SEQ
TARGET % Inhi- ID
ISIS # REGION SITE SEQUENCE bition NO.
25544 5′ UTR 30 AGAGAACCGACGGAGGAC 47 23
25545 5′ UTR 41 GTGGACTAATGAGAGAAC 0 24
25546 5′ UTR 45 GACCGTGGACTAATGAGA 40 25
25547 5′ UTR 57 AGCTGAAGACCAGACCGT 76 26
25548 5′ UTR 85 AGTCGCAAACTCGGAGAC 36 4
25549 5′ UTR 128 AATCCGAGTCCAGCCTCT 67 27
25550 5′ UTR 132 AACGAATCCGAGTCCAGC 34 28
25551 5′ UTR 138 TCAGGCAACGAATCCGAG 59 29
25552 CODING 178 CACCAACAATCACCAGTT 47 30
25553 CODING 215 AAGACTATGAGCAAGCAT 36 31
25554 CODING 243 ATACACCTCTGGGAACTG 74 32
25555 CODING 269 ACATAGTTCTCAAACACT 31 33
25556 CODING 304 ACTCTACCTGCTTTCCAT 64 34
25557 CODING 314 CACAAAGCCAACTCTACC 25 35
25558 CODING 378 AACATCGGTATCTGGGTA 35 36
25559 CODING 432 TTCTGGGATGTTTTCTAA 21 37
25560 CODING 464 GGACAGAAATGCTTGACT 64 38
25561 CODING 519 GTGCTCATCATTCCGAAG 71 39
25562 CODING 524 CTTGTGTGCTCATCATTC 38 40
25563 CODING 534 TAGCTCCCGCCTTGTGTG 78 41
25564 CODING 596 CCAATCCTGTTTGCCATA 82 42
25565 CODING 629 GTCTTTGCTGAACACTCC 56 43
25566 CODING 653 AAAACCTCTCTCACTCCA 68 44
25567 CODING 719 AAGACAAGGCAACCAGAT 55 45
25568 STOP 725 TTTCACAAGACAAGGCAA 0 46
25569 STOP 731 GCAAGGTTTCACAAGACA 37 47
25570 3′ UTR 758 ATTAACCGCATAAGGGCT 77 48
25571 3′ UTR 777 TAATAAACAGCACTTCAA 19 49
25572 3′ UTR 798 CCAGTAATCATACACTAA 26 50
25573 3′ UTR 847 ATGACTTCTGATTTGTAA 27 51
25574 3′ UTR 854 TAGCAAGATGACTTCTGA 62 52
25575 3′ UTR 858 CTGGTAGCAAGATGACTT 59 53
25576 3′ UTR 865 CTAAATACTGGTAGCAAG 29 54
25577 3′ UTR 872 TTGGCTTCTAAATACTGG 57 55
25578 3′ UTR 878 TCATAGTTGGCTTCTAAA 60 56
25579 3′ UTR 883 AATAATCATAGTTGGCTT 33 57
25580 3′ UTR 923 TCAAAAGGACCCTGGTGG 25 58
25581 3′ UTR 950 GTGCAGAGGAGGGCTGTT 68 59
25582 3′ UTR 1026 CCAACTGTTTCTCTTTCT 52 60
25583 3′ UTR 1056 AAGTAGTTACAGCCTAAT 26 61

As shown in Table 11, SEQ ID NOs 23, 26, 27, 29, 30, 32, 34, 38, 39, 41, 42, 43, 44, 45, 48, 52, 53, 56, 57, 59 and 60 demonstrated at least 45% inhibition of RhoA expression in this assay and are therefore preferred.

Example 12

Antisense Inhibition of RhoA Expression-phosphorothioate 2′-MOE gapmer Oligonucleotides

In accordance with the present invention, a second series of oligonucleotides targeted to human RhoA were synthesized. The oligonucleotide sequences are shown in Table 12. Target sites are indicated by nucleotide numbers, as given in the sequence source reference (Genbank accession no. X05026), to which the oligonucleotide binds.

All compounds in Table 12 are chimeric oligonucleotides (“gapmers”) 18 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by four-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. Cytidine residues in the 2′-MOE wings are 5-methylcytidines.

TABLE 12
Nucleotide Sequences of Human RhoA Gapmer
oligonucleotides
TARGET GENE
SEQ NUCLEOTIDE GENE
ISIS NUCLEOTIDE SEQUENCE ID CO- TARGET
NO. (5′ - >3′) NO: ORDINATES1 REGION
25584 AGAGAACCGACGGAGGAC 23 0030-0047 5′-UTR
25585 GTGGACTAATGAGAGAAC 24 0041-0058 5′-UTR
25586 GACCGTGGACTAATGAGA 25 0045-0062 5′-UTR
25587 AGCTGAAGACCAGACCGT 26 0057-0074 5′-UTR
25588 AGTCGCAAACTCGGAGAC 4 0085-0102 5′-UTR
25589 AATCCGAGTCCAGCCTCT 27 0128-0145 5′-UTR
25590 AACGAATCCGAGTCCAGC 28 0132-0149 5′-UTR
25591 TCAGGCAACGAATCCGAG 29 0138-0155 5′-UTR
25592 CACCAACAATCACCAGTT 30 0178-0195 Coding
25593 AAGACTATGAGCAAGCAT 31 0215-0232 Coding
25594 ATACACCTCTGGGAACTG 32 0243-0260 Coding
25595 ACATAGTTCTCAAACACT 33 0269-0286 Coding
25596 ACTCTACCTGCTTTCCAT 34 0304-0321 Coding
25597 CACAAAGCCAACTCTACC 35 0314-0331 Coding
25598 AACATCGGTATCTGGGTA 36 0378-0395 Coding
25599 TTCTGGGATGTTTTCTAA 37 0432-0449 Coding
25600 GGACAGAAATGCTTGACT 38 0464-0481 Coding
25601 GTGCTCATCATTCCGAAG 39 0519-0536 Coding
25602 CTTGTGTGCTCATCATTC 40 0524-0541 Coding
25603 TAGCTCCCGCCTTGTGTG 41 0534-0551 Coding
25604 CCAATCCTGTTTGCCATA 42 0596-0613 Coding
25605 GTCTTTGCTGAACACTCC 43 0629-0646 Coding
25606 AAAACCTCTCTCACTCCA 44 0653-0670 Coding
25607 AAGACAAGGCAACCAGAT 45 0719-0736 Coding
25608 TTTCACAAGACAAGGCAA 46 0725-0742 Stop
25609 GCAAGGTTTCACAAGACA 47 0731-0748 Stop
25610 ATTAACCGCATAAGGGCT 48 0758-0775 3′-UTR
25611 TAATAAACAGCACTTCAA 49 0777-0794 3′-UTR
25612 CCAGTAATCATACACTAA 50 0798-0815 3′-UTR
25613 ATGACTTCTGATTTGTAA 51 0847-0864 3′-UTR
25614 TAGCAAGATGACTTCTGA 52 0854-0871 3′-UTR
25615 CTGGTAGCAAGATGACTT 53 0858-0875 3′-UTR
25616 CTAAATACTGGTAGCAAG 54 0865-0882 3′-UTR
25617 TTGGCTTCTAAATACTGG 55 0872-0889 3′-UTR
25618 TCATAGTTGGCTTCTAAA 56 0878-0895 3′-UTR
25619 AATAATCATAGTTGGCTT 57 0883-0900 3′-UTR
25620 TCAAAAGGACCCTGGTGG 58 0923-0940 3′-UTR
25621 GTGCAGAGGAGGGCTGTT 59 0950-0967 3′-UTR
25622 CCAACTGTTTCTCTTTCT 60 1026-1043 3′-UTR
25623 AAGTAGTTACAGCCTAAT 61 1056-1073 3′-UTR
1Emboldened residues are 2′-methoxyethoxy residues (others are 2′-deoxy-). All 2′-methoxyethoxy cytidines and cytidines are 5-methyl-cytidines; all linkages are phosphorothioate linkages.
2Co-ordinates from Genbank Accession No. X05026, locus name “HSRHOB” SEQ ID NO. 1.

The oligonucleotides shown in Table 12 were tested by real-time quantitative PCR as described in other examples herein and data are shown in Table 13 (average from three experiments). If present , “N.D.” indicated “no data”.

TABLE 13
Inhibition of RhoA mRNA levels by chimeric
phosphorothioate oligonucleotides having 2′-MOE
wings and a deoxy gap
SEQ
TARGET % Inhi- ID
ISIS # REGION SITE SEQUENCE bition NO.
25584 5′ UTR 30 AGAGAACCGACGGAGGAC 44 23
25585 5′ UTR 41 GTGGACTAATGAGAGAAC 35 24
25586 5′ UTR 45 GACCGTGGACTAATGAGA 53 25
25587 5′ UTR 57 AGCTGAAGACCAGACCGT 62 26
25588 5′ UTR 85 AGTCGCAAACTCGGAGAC 54 4
25589 5′ UTR 128 AATCCGAGTCCAGCCTCT 38 27
25590 5′ UTR 132 AACGAATCCGAGTCCAGC 47 28
25591 5′ UTR 138 TCAGGCAACGAATCCGAG 31 29
25592 CODING 178 CACCAACAATCACCAGTT 0 30
25593 CODING 215 AAGACTATGAGCAAGCAT 43 31
25594 CODING 243 ATACACCTCTGGGAACTG 23 32
25595 CODING 269 ACATAGTTCTCAAACACT 16 33
25596 CODING 304 ACTCTACCTGCTTTCCAT 0 34
25597 CODING 314 CACAAAGCCAACTCTACC 0 35
25598 CODING 378 AACATCGGTATCTGGGTA 65 36
25599 CODING 432 TTCTGGGATGTTTTCTAA 53 37
25600 CODING 464 GGACAGAAATGCTTGACT 50 38
25601 CODING 519 GTGCTCATCATTCCGAAG 45 39
25602 CODING 524 CTTGTGTGCTCATCATTC 26 40
25603 CODING 534 TAGCTCCCGCCTTGTGTG 59 41
25604 CODING 596 CCAATCCTGTTTGCCATA 40 42
25605 CODING 629 GTCTTTGCTGAACACTCC 47 43
25606 CODING 653 AAAACCTCTCTCACTCCA 30 44
25607 CODING 719 AAGACAAGGCAACCAGAT 0 45
25608 STOP 725 TTTCACAAGACAAGGCAA 7 46
25609 STOP 731 GCAAGGTTTCACAAGACA 53 47
25610 3′ UTR 758 ATTAACCGCATAAGGGCT 56 48
25611 3′ UTR 777 TAATAAACAGCACTTCAA 7 49
25612 3′ UTR 798 CCAGTAATCATACACTAA 41 50
25613 3′ UTR 847 ATGACTTCTGATTTGTAA 53 51
25614 3′ UTR 854 TAGCAAGATGACTTCTGA 59 52
25615 3′ UTR 858 CTGGTAGCAAGATGACTT 67 53
25616 3′ UTR 865 CTAAATACTGGTAGCAAG 65 54
25617 3′ UTR 872 TTGGCTTCTAAATACTGG 74 55
25618 3′ UTR 878 TCATAGTTGGCTTCTAAA 52 56
25619 3′ UTR 883 AATAATCATAGTTGGCTT 49 57
25620 3′ UTR 923 TCAAAAGGACCCTGGTGG 58 58
25621 3′ UTR 950 GTGCAGAGGAGGGCTGTT 60 59
25622 3′ UTR 1026 CCAACTGTTTCTCTTTCT 62 60
25623 3′ UTR 1056 AAGTAGTTACAGCCTAAT 44 61

As shown in Table 13, SEQ ID NOs 23, 24, 25, 26, 4, 27, 28, 31, 36, 37, 38, 39, 41, 42, 43, 47, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 and 61 demonstrated at least 35% inhibition of RhoA expression in this experiment and are therefore preferred.

Example 13

Synthesis of RhoB Antisense Oligonucleotide Sequences

Oligonucleotide sequences were synthesized as described in previous examples. Antisense oligonucleotides were designed to target human RhoB. Target sequence data are from the RhoB cDNA sequence published by Chardin, P., et al. (Nucleic Acids Res., 1988, 16, 2717); Genbank accession number X06820, provided herein as SEQ ID NO: 68.

TABLE 14
Nucleotide Sequences of Human RhoB
Phosphorothioate Oligodeoxynucleotides
SEQ TARGET GENE GENE
ISIS NUCLEOTIDE SEQUENCE ID NUCLEOTIDE TARGET
NO. (5′ - >3′) NO: CO-ORDINATES1 REGION
25384 CCACCACCAGCTTCTTGC 69 0014-0031 Coding
25385 CCGTCGCCCACCACCACC 70 0024-0041 Coding
25386 GCACGTCTTGCCACACGC 71 0043-0060 Coding
25387 ACTGAACACGATCAGCAG 72 0061-0078 Coding
25388 TTACTGAACACGATCAGC 73 0063-0080 Coding
25389 CCTTACTGAACACGATCA 74 0065-0082 Coding
25390 GTCCTTACTGAACACGAT 75 0067-0084 Coding
25391 CTCGTCCTTACTGAACAC 76 0070-0087 Coding
25392 AACTCGTCCTTACTGAAC 77 0072-0089 Coding
25393 CATAGTTCTCGAAGACGG 78 0110-0127 Coding
25394 TCGGCCACATAGTTCTCG 79 0117-0134 Coding
25395 CCGTCCACCTCAATGTCG 80 0132-0149 Coding
25396 AAGCACATGAGAATGACG 81 0234-0251 Coding
25397 GAGTCCGGGCTGTCCACC 82 0255-0272 Coding
25398 ATGTTCTCCAGCGAGTCC 83 0267-0284 Coding
25399 GGGATGTTCTCCAGCGAG 84 0270-0287 Coding
25400 GACATGCTCGTCGCTGCG 85 0364-0381 Coding
25401 CGGACATGCTCGTCGCTG 86 0366-0383 Coding
25402 TGTGCGGACATGCTCGTC 87 0370-0387 Coding
25403 CTCTGTGCGGACATGCTC 88 0373-0390 Coding
25404 CCAGCTCTGTGCGGACAT 89 0377-0394 Coding
25405 CGGGCCAGCTCTGTGCGG 90 0381-0398 Coding
25406 TGCGGGCCAGCTCTGTGC 91 0383-0400 Coding
25407 GTTCCTGCTTCATGCGGG 92 0395-0412 Coding
25408 ACGGGTTCCTGCTTCATG 93 0399-0416 Coding
25409 GTAGTCGTAGGCTTGGAT 94 0451-0468 Coding
25410 CGAGGTAGTCGTAGGCTT 95 0455-0472 Coding
25411 GTCTTGGCAGAGCACTCG 96 0471-0488 Coding
25412 ACCTCGCGCACGCCTTCC 97 0492-0509 Coding
25413 AGACCTCGCGCACGCCTT 98 0494-0511 Coding
25414 CGAAGACCTCGCGCACGC 99 0497-0514 Coding
25415 CTCGAAGACCTCGCGCAC 100 0499-0516 Coding
25416 GCCGTCTCGAAGACCTCG 101 0504-0521 Coding
25417 CGTGGCCGTCTCGAAGAC 102 0508-0525 Coding
25418 GTTCTGGGAGCCGTAGCG 103 0544-0561 Coding
25419 GCCGTTCTGGGAGCCGTA 104 0547-0564 Coding
25420 GATGCAGCCGTTCTGGGA 105 0553-0570 Coding
25421 GTTGATGCAGCCGTTCTG 106 0556-0573 Coding
25422 CAGCAGTTGATGCAGCCG 107 0561-0578 Coding
25423 AGCACCTTGCAGCAGTTG 108 0570-0587 Coding
1All cytidines are 5-methyl-cytidines; all linkages are phosphorothioate linkages.
2Co-ordinates from Genbank Accession No. X06820, locus name “HSRHOB6” SEQ ID NO. 68.

Example 14

Antisense Inhibition of RhoB Expression-phosphorothioate Oligodeoxynucleotides

In accordance with the present invention, the oligonucleotides shown in Table 14 were analyzed for effect on RhoB mRNA levels by quantitative real-time PCR as described in examples herein. Data are averages from three experiments. If present, “N.D.” indicates “no data”.

TABLE 15
Inhibition of RhoB mRNA levels by phosphorothioate
oligodeoxynucleotides
SEQ
TARGET % Inhi- ID
ISIS # REGION SITE SEQUENCE bition NO.
25384 Coding 14 CCACCACCAGCTTCTTGC 0 69
25385 CODING 24 CCGTCGCCCACCACCACC 0 70
25386 CODING 43 GCACGTCTTGCCACACGC 0 71
25387 CODING 61 ACTGAACACGATCAGCAG 0 72
25388 CODING 63 TTACTGAACACGATCAGC 0 73
25389 CODING 65 CCTTACTGAACACGATCA 0 74
25390 CODING 67 GTCCTTACTGAACACGAT 5 75
25391 CODING 70 CTCGTCCTTACTGAACAC 1 76
25392 CODING 72 AACTCGTCCTTACTGAAC 30 77
25393 CODING 110 CATAGTTCTCGAAGACGG 0 78
25394 CODING 117 TCGGCCACATAGTTCTCG 13 79
25395 CODING 132 CCGTCCACCTCAATGTCG 0 80
25396 CODING 234 AAGCACATGAGAATGACG 0 81
25397 CODING 255 GAGTCCGGGCTGTCCACC 0 82
25398 CODING 267 ATGTTCTCCAGCGAGTCC 0 83
25399 CODING 270 GGGATGTTCTCCAGCGAG 33 84
25400 CODING 364 GACATGCTCGTCGCTGCG 0 85
25401 CODING 366 CGGACATGCTCGTCGCTG 0 86
25402 CODING 370 TGTGCGGACATGCTCGTC 0 87
25403 CODING 373 CTCTGTGCGGACATGCTC 39 88
25404 CODING 377 CCAGCTCTGTGCGGACAT 21 89
25405 CODING 381 CGGGCCAGCTCTGTGCGG 38 90
25406 CODING 383 TGCGGGCCAGCTCTGTGC 31 91
25407 CODING 395 GTTCCTGCTTCATGCGGG 27 92
25408 CODING 399 ACGGGTTCCTGCTTCATG 0 93
25409 CODING 451 GTAGTCGTAGGCTTGGAT 29 94
25410 CODING 455 CGAGGTAGTCGTAGGCTT 39 95
25411 CODING 471 GTCTTGGCAGAGCACTCG 20 96
25412 CODING 492 ACCTCGCGCACGCCTTCC 0 97
25413 CODING 494 AGACCTCGCGCACGCCTT 16 98
25414 CODING 497 CGAAGACCTCGCGCACGC 0 99
25415 CODING 499 CTCGAAGACCTCGCGCAC 0 100
25416 CODING 504 GCCGTCTCGAAGACCTCG 0 101
25417 CODING 508 CGTGGCCGTCTCGAAGAC 0 102
25418 CODING 544 GTTCTGGGAGCCGTAGCG 36 103
25419 CODING 547 GCCGTTCTGGGAGCCGTA 0 104
25420 CODING 553 GATGCAGCCGTTCTGGGA 0 105
25421 CODING 556 GTTGATGCAGCCGTTCTG 7 106
25422 CODING 561 CAGCAGTTGATGCAGCCG 31 107
25423 CODING 570 AGCACCTTGCAGCAGTTG 0 108

As shown in Table 15, SEQ ID Nos 77, 84, 88, 90, 91, 92, 94, 95, 103 and 107 demonstrated at least 25% inhibition of RhoB expression in this assay and are therefore preferred.

Example 15

Antisense Inhibition of RhoB Expression-phosphorothioate 2′-MOE gapmer Oligonucleotides

In accordance with the present invention, a second series oligonucleotides targeted to human RhoB were synthesized. The oligonucleotide sequences are shown in Table 16. Target sites are indicated by nucleotide numbers, as given in the sequence source reference (Genbank accession no. X06820), to which the oligonucleotide binds.

All compounds in Table 16 are chimeric oligonucleotides (“gapmers”) 18 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by four-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. Cytidine residues in the 2′-MOE wings are 5-methylcytidines.

TABLE 16
Nucleotide Sequences of Human RhoB Gapiner
Oligonucleotides
SEQ TARGET GENE GENE
ISIS NUCLEOTIDE SEQUENCE ID NUCLEOTIDE TARGET
NO. (51 ->31) NO: CO-ORDINATES1 REGION
25424 CCACCACCAGCTTCTTGC 69 0014-0031 Coding
25425 CCGTCGCCCACCACCACC 70 0024-0041 Coding
25426 GCACGTCTTGCCACACGC 71 0043-0060 Coding
25427 ACTGAACACGATCAGCAG 72 0061-0078 Coding
25428 TTACTGAACACGATCAGC 73 0063-0080 Coding
25429 CCTTACTGAACACGATCA 74 0065-0082 Coding
25430 GTCCTTACTGAACACGAT 75 0067-0084 Coding
25431 CTCGTCCTTACTGAACAC 76 0070-0087 Coding
25432 AACTCGTCCTTACTGAAC 77 0072-0089 Coding
25433 CATAGTTCTCGAAGACGG 78 0110-0127 Coding
25434 TCGGCCACATAGTTCTCG 79 0117-0134 Coding
25435 CCGTCCACCTCAATGTCG 80 0132-0149 Coding
25436 AAGCACATGAGAATGACG 81 0234-0251 Coding
25437 GAGTCCGGGCTGTCCACC 82 0255-0272 Coding
25438 ATGTTCTCCAGCGAGTCC 83 0267-0284 Coding
25439 GGGATGTTCTCCAGCGAG 84 0270-0287 Coding
25440 GACATGCTCGTCGCTGCG 85 0364-0381 Coding
25441 CGGACATGCTCGTCGCTG 86 0366-0383 Coding
25442 TGTGCGGACATGCTCGTC 87 0370-0387 Coding
25443 CTCTGTGCGGACATGCTC 88 0373-0390 Coding
25444 CCAGCTCTGTGCGGACAT 89 0377-0394 Coding
25445 CGGGCCAGCTCTGTGCGG 90 0381-0398 Coding
25446 TGCGGGCCAGCTCTGTGC 91 0383-0400 Coding
25447 GTTCCTGCTTCATGCGGG 92 0395-0412 Coding
25448 ACGGGTTCCTGCTTCATG 93 0399-0416 Coding
25449 GTAGTCGTAGGCTTGGAT 94 0451-0468 Coding
25450 CGAGGTAGTCGTAGGCTT 95 0455-0472 Coding
25451 GTCTTGGCAGAGCACTCG 96 0471-0488 Coding
25452 ACCTCGCGCACGCCTTCC 97 0492-0509 Coding
25453 AGACCTCGCGCACGCCTT 98 0494-0511 Coding
25454 CGAAGACCTCGCGCACGC 99 0497-0514 Coding
25455 CTCGAAGACCTCGCGCAC 100 0499-0516 Coding
25456 GCCGTCTCGAAGACCTCG 101 0504-0521 Coding
25457 CGTGGCCGTCTCGAAGAC 102 0508-0525 Coding
25458 GTTCTGGGAGCCGTAGCG 103 0544-0561 Coding
25459 GCCGTTCTGGGAGCCGTA 104 0547-0564 Coding
25460 GATGCAGCCGTTCTGGGA 105 0553-0570 Coding
25461 GTTGATGCAGCCGTTCTG 106 0556-0573 Coding
25462 CAGCAGTTGATGCAGCCG 107 0561-0578 Coding
25463 AGCACCTTGCAGCAGTTG 108 0570-0587 Coding
1Emboldened residues are 2′-methaxyethoxy residues (others are 2′-deoxy-). A11 2′-methoxyethoxy cytidines and cytidines are 5-methyl-cytidines; all linkages are phosphorothioate linkages.
2Co-ordinates from Genbank Accession No. X06820, locus name ″HSRHOB6″ SEQ ID NO. 68.

Data for the compounds in Table 16 were obtained by real-time quantitative PCR as described in other examples herein and are averaged from three experiments. Results are shown in Table 17. If present, “N.D.” indicates “no data”.

TABLE 17
Inhibition of RhoB mRNA levels by chimeric
phosphorothioate oligonucleotides having 2′-MOE
wings and a deoxy gap
SEQ
TARGET % Inhi- ID
ISIS # REGION SITE SEQUENCE bition NO.
25424 Coding 14 CCACCACCAGCTTCTTGC 29 69
25425 CODING 24 CCGTCGCCCACCACCACC 23 70
25426 CODING 43 GCACGTCTTGCCACACGC 46 71
25427 CODING 61 ACTGAACACGATCAGCAG 37 72
25428 CODING 63 TTACTGAACACGATCAGC 47 73
25429 CODING 65 CCTTACTGAACACGATCA 7 74
25430 CODING 67 GTCCTTACTGAACACGAT 46 75
25431 CODING 70 CTCGTCCTTACTGAACAC 52 76
25432 CODING 72 AACTCGTCCTTACTGAAC 35 77
25433 CODING 110 CATAGTTCTCGAAGACGG 29 78
25434 CODING 117 TCGGCCACATAGTTCTCG 65 79
25435 CODING 132 CCGTCCACCTCAATGTCG 40 80
25436 CODING 234 AAGCACATGAGAATGACG 44 81
25437 CODING 255 GAGTCCGGGCTGTCCACC 36 82
25438 CODING 267 ATGTTCTCCAGCGAGTCC 28 83
25439 CODING 270 GGGATGTTCTCCAGCGAG 54 84
25440 CODING 364 GACATGCTCGTCGCTGCG 49 85
25441 CODING 366 CGGACATGCTCGTCGCTG 46 86
25442 CODING 370 TGTGCGGACATGCTCGTC 65 87
25443 CODING 373 CTCTGTGCGGACATGCTC 39 88
25444 CODING 377 CCAGCTCTGTGCGGACAT 19 89
25445 CODING 381 CGGGCCAGCTCTGTGCGG 21 90
25446 CODING 383 TGCGGGCCAGCTCTGTGC 9 91
25447 CODING 395 GTTCCTGCTTCATGCGGG 16 92
25448 CODING 399 ACGGGTTCCTGCTTCATG 7 93
25449 CODING 451 GTAGTCGTAGGCTTGGAT 38 94
25450 CODING 455 CGAGGTAGTCGTAGGCTT 0 95
25451 CODING 471 GTCTTGGCAGAGCACTCG 42 96
25452 CODING 492 ACCTCGCGCACGCCTTCC 9 97
25453 CODING 494 AGACCTCGCGCACGCCTT 7 98
25454 CODING 497 CGAAGACCTCGCGCACGC 12 99
25455 CODING 499 CTCGAAGACCTCGCGCAC 23 100
25456 CODING 504 GCCGTCTCGAAGACCTCG 34 101
25457 CODING 508 CGTGGCCGTCTCGAAGAC 27 102
25458 CODING 544 GTTCTGGGAGCCGTAGCG 58 103
25459 CODING 547 GCCGTTCTGGGAGCCGTA 63 104
25460 CODING 553 GATGCAGCCGTTCTGGGA 17 105
25461 CODING 556 GTTGATGCAGCCGTTCTG 21 106
25462 CODING 561 CAGCAGTTGATGCAGCCG 50 107
25463 CODING 570 AGCACCTTGCAGCAGTTG 55 108

As shown in Table 17, SEQ ID Nos 71, 62, 63, 75, 76, 77, 79, 80, 81, 82, 84, 85, 86, 87, 88, 94, 96, 101, 103, 104, 107 and 108 demonstrated at least 30% inhibition of RhoB expression in this experiment and are therefore preferred.

Example 16

Synthesis of RhoC Antisense Oligonucleotide Sequences

Oligonucleotide sequences were synthesized as described in previous examples. Antisense oligonucleotides were designed to target human RhoC. Target sequence data are from the RhoC cDNA sequence determined by Fagan, K. P., et al.; Genbank accession number L25081, provided herein as SEQ ID NO: 109.

TABLE 18
Nucleotide Sequences of Human RhoC Phosphorothioate
Oligonucleotides
SEQ TARGET GENE GENE
ISIS NUCLEOTIDE SEQUENCE ID NUCLEOTIDE TARGET
NO. (5′->3′) NO: CO-ORDINATES1 REGION
25304 GAGCTGAGATGAAGTCAA 110 0004-0021 5′-UTR
25305 GCTGAAGTTCCCAGGCTG 111 0044-0061 5′-UTR
25306 CCGGCTGAAGTTCCCAGG 112 0047-0064 5′-UTR
25307 GGCACCATCCCCAACGAT 113 0104-0121 Coding
25308 AGGCACCATCCCCAACGA 114 0105-0122 Coding
25309 TCCCACAGGCACCATCCC 115 0111-0128 Coding
25310 AGGTCTTCCCACAGGCAC 116 0117-0134 Coding
25311 ATGAGGAGGCAGGTCTTC 117 0127-0144 Coding
25312 TTGCTGAAGACGATGAGG 118 0139-0156 Coding
25313 TCAAAGACAGTAGGGACG 119 0178-0195 Coding
25314 TTCTCAAAGACAGTAGGG 120 0181-0198 Coding
25315 AGTTCTCAAAGACAGTAG 121 0183-0200 Coding
25316 TGTTTTCCAGGCTGTCAG 122 0342-0359 Coding
25317 TCGTCTTGCCTCAGGTCC 123 0433-0450 Coding
25318 GTGTGCTCGTCTTGCCTC 124 0439-0456 Coding
25319 CTCCTGGTGTGCTCGTCT 125 0445-0462 Coding
25320 CAGACCGAACGGGCTCCT 126 0483-0500 Coding
25321 TTCCTCAGACCGAACGGG 127 0488-0505 Coding
25322 ACTCAAGGTAGCCAAAGG 128 0534-0551 Coding
25323 CTCCCGCACTCCCTCCTT 129 0566-0583 Coding
25324 CTCAAACACCTCCCGCAC 130 0575-0592 Coding
25325 GGCCATCTCAAACACCTC 131 0581-0598 Coding
25326 CTTGTTCTTGCGGACCTG 132 0614-0631 Coding
25327 CCCCTCCGACGCTTGTTC 133 0625-0642 Coding
25328 GTATGGAGCCCTCAGGAG 134 0737-0754 3′-UTR
25329 GAGCCTTCAGTATGGAGC 135 0746-0763 3′-UTR
25330 GAAAATGGAGCCTTCAGT 136 0753-0770 3′-UTR
25331 GGAACTGAAAATGGAGCC 137 0759-0776 3′-UTR
25332 GGAGGGAACTGAAAATGG 138 0763-0780 3′-UTR
25333 GCAGGAGGGAACTGAAAA 139 0766-0783 3′-UTR
25334 AGGGCAGGGCATAGGCGT 140 0851-0868 3′-UTR
25335 GGAAGGGCAGGGCATAGG 141 0854-0871 3′-UTR
25336 CATGAGGAAGGGCAGGGC 142 0859-0876 3′-UTR
25337 TAAAGTGCTGGTGTGTGA 143 0920-0937 3′-UTR
25338 CCTGTGAGCCAGAAGTGT 144 0939-0956 3′-UTR
25339 TTCCTGTGAGCCAGAAGT 145 0941-0958 3′-UTR
25340 CACTTTCCTGTGAGCCAG 146 0945-0962 3′-UTR
25341 AGACACTTTCCTGTGAGC 147 0948-0965 3′-UTR
25342 ACTCTGGGTCCCTACTGC 148 0966-0983 3′-UTR
25343 TGCAGAAACAACTCCAGG 149 0992-1009 3′-UTR
1A11 cytidines are 5-methyl-cytidines; all linkages are phosphorothioate linkages.
2Co-ordinates from Genbank Accession No. L25081, locus name
″HUMRHOCA″ SEQ ID NO. 109.

The compounds shown in Table 18 were analyzed for effect on RhoC mRNA levels by quantitative real-time PCR as described in examples herein. Data are shown in Table 19 and are averages from three experiments. If present, “N.D.” indicates “no data”.

TABLE 19
Inhibition of RhoC mRNA levels by phosphorothioate
oligodeoxynucleotides
SEQ
TARGET % Inhi- ID
ISIS # REGION SITE SEQUENCE bition NO
25304 5′UTR 4 GAGCTGAGATGAAGTCAA 29 110
25305 5′UTR 44 GCTGAAGTTCCCAGGCTG 25 111
25306 5′UTR 47 CCGGCTGAAGTTCCCAGG 42 112
25307 CODING 104 GGCACCATCCCCAACGAT 81 113
25308 CODING 105 AGGCACCATCCCCAACGA 81 114
25309 CODING 111 TCCCACAGGCACCATCCC 70 115
25310 CODING 117 AGGTCTTCCCACAGGCAC 40 116
25311 CODING 127 ATGAGGAGGCAGGTCTTC 41 117
25312 CODING 139 TTGCTGAAGACGATGAGG 23 118
25313 CODING 178 TCAAAGACAGTAGGGACG 0 119
25314 CODING 181 TTCTCAAAGACAGTAGGG 2 120
25315 CODING 183 AGTTCTCAAAGACAGTAG 38 121
25316 CODING 342 TGTTTTCCAGGCTGTCAG 59 122
25317 CODING 433 TCGTCTTGCCTCAGGTCC 79 123
25318 CODING 439 GTGTGCTCGTCTTGCCTC 67 124
25319 CODING 445 CTCCTGGTGTGCTCGTCT 67 125
25320 CODING 483 CAGACCGAACGGGCTCCT 65 126
25321 CODING 488 TTCCTCAGACCGAACGGG 57 127
25322 CODING 534 ACTCAAGGTAGCCAAAGG 33 128
25323 CODING 566 CTCCCGCACTCCCTCCTT 91 129
25324 CODING 575 CTCAAACACCTCCCGCAC 34 130
25325 CODING 581 GGCCATCTCAAACACCTC 64 131
25326 CODING 614 CTTGTTCTTGCGGACCTG 72 132
25327 CODING 625 CCCCTCCGACGCTTGTTC 66 133
25328 3′UTR 737 GTATGGAGCCCTCAGGAG 60 134
25329 3′UTR 746 GAGCCTTCAGTATGGAGC 63 135
25330 3′UTR 753 GAAAATGGAGCCTTCAGT 24 136
25331 3′UTR 759 GGAACTGAAAATGGAGCC 2 137
25332 3′UTR 763 GGAGGGAACTGAAAATGG 13 138
25333 3′UTR 766 GCAGGAGGGAACTGAAAA 27 139
25334 3′UTR 851 AGGGCAGGGCATAGGCGT 31 140
25335 3′UTR 854 GGAAGGGCAGGGCATAGG 21 141
25336 3′UTR 859 CATGAGGAAGGGCAGGGC 0 142
25337 3′UTR 920 TAAAGTGCTGGTGTGTGA 39 143
25338 3′UTR 939 CCTGTGAGCCAGAAGTGT 69 144
25339 3′UTR 941 TTCCTGTGAGCCAGAAGT 69 145
25340 3′UTR 945 CACTTTCCTGTGAGCCAG 82 146
25341 3′UTR 948 AGACACTTTCCTGTGAGC 69 147
25342 3′UTR 966 ACTCTGGGTCCCTACTGC 20 148
25343 3′UTR 992 TGCAGAAACAACTCCAGG 0 149

As shown in Table 19, SEQ ID NOs 113, 114, 115, 122, 123, 124, 125, 126, 127, 129, 131, 132, 133, 134, 135, 144, 145, 146 and 147 demonstrated at least 50% inhibition of RhoC expression in this assay and are therefore preferred.

Example 17

Antisense Inhibition of RhoC Expression-phosphorothioate 2′-MOE Gapmer Oligonucleotides

In accordance with the present invention, a second series of oligonucleotides targeted to human RhoC were synthesized. The oligonucleotide sequences are shown in Table 20. Target sites are indicated by nucleotide numbers, as given in the sequence source reference (Genbank accession no. L25081), to which the oligonucleotide binds.

All compounds in Table 20 are chimeric oligonucleotides (“gapmers”) 18 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by four-nucleotide “wings”. The wings are composed of 21-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. Cytidine residues in the 2′-MOE wings are 5-methylcytidines.

TABLE 20
Nucleotide Sequences of Human RhoC Gapmer
Oligonucleotides
SEQ TARGET GENE GENE
ISIS NUCLEOTIDE SEQUENCE ID NUCLEOTIDE TARGET
NO. (5′->3′) NO: CO-ORDINATES1 REGION
25344 GAGCTGAGATGAAGTCAA 110 0004-0021 5′-UTR
25345 GCTGAAGTTCCCAGGCTG 111 0044-0061 5′-UTR
25346 CCGGCTGAAGTTCCCAGG 112 0047-0064 5′-UTR
25347 GGCACCATCCCCAACGAT 113 0104-0121 Coding
25348 AGGCACCATCCCCAACGA 114 0105-0122 Coding
25349 TCCCACAGGCACCATCCC 115 0111-0128 Coding
25350 AGGTCTTCCCACAGGCAC 116 0117-0134 Coding
25351 ATGAGGAGGCAGGTCTTC 117 0127-0144 Coding
25352 TTGCTGAAGACGATGAGG 118 0139-0156 Coding
25353 TCAAAGACAGTAGGGACG 119 0178-0195 Coding
25354 TTCTCAAAGACAGTAGGG 120 0181-0198 Coding
25355 AGTTCTCAAAGACAGTAG 121 0183-0200 Coding
25356 TGTTTTCCAGGCTGTCAG 122 0342-0359 Coding
25357 TCGTCTTGCCTCAGGTCC 123 0433-0450 Coding
25358 GTGTGCTCGTCTTGCCTC 124 0439-0456 Coding
25359 CTCCTGGTGTGCTCGTCT 125 0445-0462 Coding
25360 CAGACCGAACGGGCTCCT 126 0483-0500 Coding
25361 TTCCTCAGACCGAACGGG 127 0488-0505 Coding
25362 ACTCAAGGTAGCCAAAGG 128 0534-0551 Coding
25363 CTCCCGCACTCCCTCCTT 129 0566-0583 Coding
25364 CTCAAACACCTCCCGCAC 130 0575-0592 Coding
25365 GGCCATCTCAAACACCTC 131 0581-0598 Coding
25366 CTTGTTCTTGCGGACCTG 132 0614-0631 Coding
25367 CCCCTCCGACGCTTGTTC 133 0625-0642 Coding
25368 GTATGGAGCCCTCAGGAG 134 0737-0754 3′-UTR
25369 GAGCCTTCAGTATGGAGC 135 0746-0763 3′-UTR
25370 GAAAATGGAGCCTTCAGT 136 0753-0770 3′-UTR
25371 GGAACTGAAAATGGAGCC 137 0759-0776 3′-UTR
25372 GGAGGGAACTGAAAATGG 138 0763-0780 3′-UTR
25373 GCAGGAGGGAACTGAAAA 139 0766-0783 3′-UTR
25374 AGGGCAGGGCATAGGCGT 140 0851-0868 3′-UTR
25375 GGAAGGGCAGGGCATAGG 141 0854-0871 3′-UTR
25376 CATGAGGAAGGGCAGGGC 142 0859-0876 3′-UTR
25377 TAAAGTGCTGGTGTGTGA 143 0920-0937 3′-UTR
25378 CCTGTGAGCCAGAAGTGT 144 0939-0956 3′-UTR
25379 TTCCTGTGAGCCAGAAGT 145 0941-0958 3′-UTR
25380 CACTTTCCTGTGAGCCAG 146 0945-0962 3′-UTR
25381 AGACACTTTCCTGTGAGC 147 0948-0965 3′-UTR
25382 ACTCTGGGTCCCTACTGC 148 0966-0983 3′-UTR
25383 TGCAGAAACAACTCCAGG 149 0992-1009 3′-UTR
1Emboldened residues are 2′-methoxyethoxy residues (others are 2′-deoxy-). All 2′-methoxyethoxy cytidines and cytidines are 5-methyl-cytidines; all linkages are phosphorothioate linkages.
2Co-ordinates from Genbank Accession No. L25081, locus name ″HUMRHOCA″ SEQ ID NO. 109.

RhoC inhibition data for these compounds were obtained by real-time quantitative PCR as described in other examples herein and are averaged from three experiments. Data are shown in Table 21. If present, “N.D.” indicates “no data”.

TABLE 21
Inhibition of RhoC mRNA levels by chimeric
phosphorothioate oligonucleotides having
2′-MOE wings and a deoxy gap
SEQ
TARGET % Inhi- ID
ISIS# REGION SITE SEQUENCE bition NO.
25344 5′UTR 4 GAGCTGAGATGAAGTCAA 0 110
25345 5′UTR 44 GCTGAAGTTCCCAGGCTG 35 111
25346 5′UTR 47 CCGGCTGAAGTTCCCAGG 53 112
25347 Coding 104 GGCACCATCCCCAACGAT 50 113
25348 Coding 105 AGGCACCATCCCCAACGA 56 114
25349 Coding 111 TCCCACAGGCACCATCCC 4 115
25350 Coding 117 AGGTCTTCCCACAGGCAC 11 116
25351 Coding 127 ATGAGGAGGCAGGTCTTC 6 117
25352 Coding 139 TTGCTGAAGACGATGAGG 15 118
25353 Coding 178 TCAAAGACAGTAGGGACG 32 119
25354 Coding 181 TTCTCAAAGACAGTAGGG 7 120
25355 Coding 183 AGTTCTCAAAGACAGTAG 39 121
25356 Coding 342 TGTTTTCCAGGCTGTCAG 59 122
25357 Coding 433 TCGTCTTGCCTCAGGTCC 48 123
25358 Coding 439 GTGTGCTCGTCTTGCCTC 36 124
25359 Coding 445 CTCCTGGTGTGCTCGTCT 61 125
25360 Coding 483 CAGACCGAACGGGCTCCT 50 126
25361 Coding 488 TTCCTCAGACCGAACGGG 14 127
25362 Coding 534 ACTCAAGGTAGCCAAAGG 32 128
25363 Coding 566 CTCCCGCACTCCCTCCTT 21 129
25364 Coding 575 CTCAAACACCTCCCGCAC 9 130
25365 Coding 581 GGCCATCTCAAACACCTC 66 131
25366 Coding 614 CTTGTTCTTGCGGACCTG 61 132
25367 Coding 625 CCCCTCCGACGCTTGTTC 0 133
25368 3′UTR 737 GTATGGAGCCCTCAGGAG 28 134
25369 3′UTR 746 GAGCCTTCAGTATGGAGC 32 135
25370 3′UTR 753 GAAAATGGAGCCTTCAGT 0 136
25371 3′UTR 759 GGAACTGAAAATGGAGCC 40 137
25372 3′UTR 763 GGAGGGAACTGAAAATGG 45 138
25373 3′UTR 766 GCAGGAGGGAACTGAAAA 35 139
25374 3′UTR 851 AGGGCAGGGCATAGGCGT 5 140
25375 3′UTR 854 GGAAGGGCAGGGCATAGG 0 141
25376 3′UTR 859 CATGAGGAAGGGCAGGGC 0 142
25377 3′UTR 920 TAAAGTGCTGGTGTGTGA 20 143
25378 3′UTR 939 CCTGTGAGCCAGAAGTGT 67 144
25379 3′UTR 941 TTCCTGTGAGCCAGAAGT 61 145
25380 3′UTR 945 CACTTTCCTGTGAGCCAG 80 146
25381 3′UTR 948 AGCAACTTTCCTGTGAGC 0 147
25382 3′UTR 966 ACTCTGGGTCCCTACTGC 0 148
25383 3′UTR 992 TGCAGAAACAACTCCAGG 0 149

As shown in Table 21, SEQ ID NOs 111, 112, 113, 114, 119, 121, 122, 123, 124, 125, 126, 128, 131, 132, 134, 135, 137, 138, 139, 144, 145 and 146 demonstrated at least 251 inhibition of RhoC expression in this experiment and are therefore preferred.

Example 18

Synthesis of RhoG Antisense Oligonucleotide Sequences

Oligonucleotide sequences designed to target human RhoG were synthesized as described in previous examples and are shown in Table 22. Target sequence data are from the RhoG cDNA sequence published by Vincent, S., et al. (Mol. Cell. Biol. 1992, 12, 3138-3148); Genbank accession number X61587, provided herein as SEQ ID NO: 150.

TABLE 22
Nucleotide Sequences of Human RhoG
Phosphorothioate Oligodeoxynucleotide
SEQ TARGET GENE GENE
ISIS NUCLEOTIDE SEQUENCE ID NUCLEOTIDE TARGET
NO. (5′ -> 3′) NO: CO-ORDINATES1 REGION
25464 GACCTGGTGCCCCTCCCG 151 0048-0065 5′-UTR
25465 TCTTCTGGACCCCTCTGG 152 0073-0090 5′-UTR
25466 GGCAGTGCCTCCTCTCTC 153 0089-0106 5′-UTR
25467 GTGCAGTTGCTGTAGTGA 154 0107-0124 5′-UTR
25468 GCATCGTGGGTGCAGTTG 155 0116-0133 AUG
25469 CCACCACGCACTTGATGC 156 0137-0154 Coding
25470 TTGTGTAGCAGATGAGCA 157 0185-0202 Coding
25471 AAAGCGTTAGTTGTGTAG 158 0195-0212 Coding
25472 GCGCGCTGTAATTGTCGA 159 0239-0256 Coding
25473 GGTTCACTGTGCGCCCGT 160 0269-0286 Coding
25474 GTCCCACAGGTTCAGGTT 161 0283-0300 Coding
25475 TGTACGGAGGCGGTCATA 162 0319-0336 Coding
25476 ACGTTGGTCTGAGGGTAG 163 0342-0359 Coding
25477 CAATGGAGAAACAGATGA 164 0365-0382 Coding
25478 CATAGGACGGCGGACTGG 165 0383-0400 Coding
25479 CGCACGTTCTCATAGGAC 166 0393-0410 Coding
25480 ACCTCTGGATGCCACTTG 167 0414-0431 Coding
25481 AGGGCAGTGGTGGCACAC 168 0430-0447 Coding
25482 CAGCAGGATGGGCACATC 169 0448-0465 Coding
25483 GGGTGTCAGGCTGGGCTC 170 0488-0505 Coding
25484 CCCTGCTGCGGTGTGATG 171 0537-0554 Coding
25485 CGCGAGTGCCTGGCCCTG 172 0550-0567 Coding
25486 GTAGCGCACAGCGTGGAT 173 0574-0591 Coding
25487 CATTCGAGGTAGCGCACA 174 0582-0599 Coding
25488 ACACCATCCTGTTGCAGG 175 0606-0623 Coding
25489 GAACACTTCCTTGACACC 176 0619-0636 Coding
25490 ACAGCCTCGGCGAACACT 177 0630-0647 Coding
25491 AAGAGGATGCAGGACCGC 178 0684-0701 Coding
25492 GCAGCCTCCAAGCCAAGT 179 0713-0730 3′-UTR
25493 AAAAGGCATTCAGGGAAC 180 0818-0835 3′-UTR
25494 GGGTCCAACCTTGGCTTG 181 0936-0953 3′-UTR
25495 GTCAGTAGCGGAAAATGG 182 0984-1001 3′-UTR
25496 AGCTGGATGAACTGGTCA 183 0998-1015 3′-UTR
25497 AACTGTGTGGAAAGCTGG 184 1010-1027 3′-UTR
25498 ACCACAATAGGCAGCAAC 185 1028-1045 3′-UTR
25499 GAGGGCAGAGGTTAGAGA 186 1074-1091 3′-UTR
25500 CAATTCCAAGAGCAGCGA 187 1090-1107 3′-UTR
25501 TGGAGAAGGGAGAGAGCA 188 1119-1136 3′-UTR
25502 ACATTCACCTTCTCAGGA 189 1154-1171 3′-UTR
25503 GTCAGCAAATGCGTAAGG 190 1199-1216 3′-UTR
1All cytidines are 5-methyl-cytidines; all linkages are phosphorothioate linkages.
2Co-ordinates from Genbank Accession No. X61587, locus name “HSRHOG” SEQ ID NO. 150.

The compounds in Table 22 were analyzed for effect on RhoG mRNA levels by quantitative real-time PCR as described in other examples herein. Data, shown in Table 23, are averages from three experiments. If present, “N.D.” indicates “no data”.

TABLE 23
Inhibition of RhoG mRNA levels by
phosphorothioate Oligodeoxynucleotides
SEQ
TARGET % Inhi- ID
ISIS# REGION SITE SEQUENCE bition NO.
25464 5′ UTR 48 GACCTGGTGCCCCTCCCG 35 151
25465 5′ UTR 73 TCTTCTGGACCCCTCTGG 36 152
25466 5′ UTR 89 GGCAGTGCCTCCTCTCTC 35 153
25467 5′ UTR 107 GTGCAGTTGCTGTAGTGA 10 154
25468 5′ UTR 116 GCATCGTGGGTGCAGTTG 47 155
25469 CODING 137 CCACCACGCACTTGATGC 14 156
25470 CODING 185 TTGTGTAGCAGATGAGCA 35 157
25471 CODING 195 AAAGCGTTAGTTGTGTAG 0 158
25472 CODING 239 GCGCGCTGTAATTGTCGA 36 159
25473 CODING 269 GGTTCACTGTGCGCCCGT 16 160
25474 CODING 283 GTCCCACAGGTTCAGGTT 31 161
25475 CODING 319 TGTACGGAGGCGGTCATA 37 162
25476 CODING 342 ACGTTGGTCTGAGGGTAG 38 163
25477 CODING 365 CAATGGAGAAACAGATGA 0 164
25478 CODING 383 CATAGGACGGCGGACTGG 17 165
25479 CODING 393 CGCACGTTCTCATAGGAC 24 166
25480 CODING 414 ACCTCTGGATGCCACTTG 35 167
25481 CODING 430 AGGGCAGTGGTGGCACAC 15 168
25482 CODING 448 CAGCAGGATGGGCACATC 20 169
25483 CODING 488 GGGTGTCAGGCTGGGCTC 15 170
25484 CODING 537 CCCTGCTGCGGTGTGATG 44 171
25464 5′ UTR 48 GACCTGGTGCCCCTCCCG 35 151
25465 5′ UTR 73 TCTTCTGGACCCCTCTGG 36 152
25466 5′ UTR 89 GGCAGTGCCTCCTCTCTC 35 153
25485 CODING 550 CGCGAGTGCCTGGCCCTG 9 172
25486 CODING 574 GTAGCGCACAGCGTGGAT 35 173
25487 CODING 582 CATTCGAGGTAGCGCACA 39 174
25488 CODING 606 ACACCATCCTGTTGCAGG 23 175
25489 CODING 619 GAACACTTCCTTGACACC 31 176
25490 CODING 630 ACAGCCTCGGCGAACACT 6 177
25491 CODING 684 AAGAGGATGCAGGACCGC 18 178
25492 3′ UTR 713 GCAGCCTCCAAGCCAAGT 42 179
25493 3′ UTR 818 AAAAGGCATTCAGGGAAC 0 180
25494 3′ UTR 936 GGGTCCAACCTTGGCTTG 58 181
25495 3′ UTR 984 GTCAGTAGCGGAAAATGG 0 182
25496 3′ UTR 998 AGCTGGATGAACTGGTCA 23 183
25497 3′ UTR 1010 AACTGTGTGGAAAGCTGG 8 184
25498 3′ UTR 1028 ACCACAATAGGCAGCAAC 31 185
25499 3′ UTR 1074 GAGGGCAGAGGTTAGAGA 21 186
25500 3′ UTR 1090 CAATTCCAAGAGCAGCGA 18 187
25501 3′ UTR 1119 TGGAGAAGGGAGAGAGCA 32 188
25502 3′ UTR 1154 ACATTCACCTTCTCAGGA 20 189
25503 3′ UTR 1199 GTCAGCAAATGCGTAAGG 24 190

As shown in Table 23, SEQ ID NOs 151, 152, 153, 155, 157, 159, 61, 162, 163, 167, 171, 173, 174, 176, 179, 181, 185 and 188 demonstrated at least 25% inhibition of RhoG expression in this assay and are therefore preferred.

Example 19

Antisense Inhibition of RhoG Expression-phosphorothioate 2′-MOE Gapmer Oligonucleotides

In accordance with the present invention, a second series of oligonucleotides targeted to human RhoG were synthesized. The oligonucleotide sequences are shown in Table 24. Target sites are indicated by nucleotide numbers, as given in the sequence source reference (Genbank accession no. X61587), to which the oligonucleotide binds.

All compounds in Table 24 are chimeric oligonucleotides (“gapmers”) 18 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by four-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOB)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. Cytidine residues in the 2′-MOE wings are 5-methylcytidines.

TABLE 24
Nucleotide Sequences of
Human RhoG Gapmer Oligonucleotides
SEQ TARGET GENE GENE
ISIS NUCLEOTIDE SEQUENCE ID NUCLEOTIDE TARGET
NO. (5′ -> 3′) NO: CO-ORDINATES1 REGION
25504 GACCTGGTGCCCCTCCCG 151 0048-0065 5′-UTR
25505 TCTTCTGGACCCCTCTGG 152 0073-0090 5′-UTR
25506 GGCAGTGCCTCCTCTCTC 153 0089-0106 5′-UTR
25507 GTGCAGTTGCTGTAGTGA 154 0107-0124 5′-UTR
25508 GCATCGTCCOTGCAGTTG 155 0116-0133 AUG
25509 CCACCACGCACTTGATGC 156 0137-0154 Coding
25510 TTGTGTAGCAGATGAGCA 157 0185-0202 Coding
25511 AAAGCGTTAGTTGTGTAG 158 0195-0212 Coding
25512 GCGCGCTGTAATTGTCGA 159 0239-0256 Coding
25513 GGTTCACTGTGCGCCCGT 160 0269-0286 Coding
25514 GTCCCACAGGTTCAGGTT 161 0283-0300 Coding
25515 TGTACGGAGGCGGTCATA 162 0319-0336 Coding
25516 ACGTTGGTCTGAGGGTAG 163 0342-0359 Coding
25517 CAATGGAGAAACAGATGA 164 0365-0382 Coding
25518 CATAGGACGGCGGACTGG 165 0383-0400 Coding
25519 CGCACGTTCTCATAGGAC 166 0393-0410 Coding
25520 ACCTCTGGATGCCACTTG 167 0414-0431 Coding
25521 AGGGCAGTGGTGGCACAC 168 0430-0447 Coding
25522 CAGCAGGATGGGCACATC 169 0448-0465 Coding
25523 GGGTGTCAGGCTGGGCTC 170 0488-0505 Coding
25524 CCCTGCTGCGGTGTGATG 171 0537-0554 Coding
25525 CGCGAGTGCCTGGCCCTG 172 0550-0567 Coding
25526 GTAGCGCACAGCGTGGAT 173 0574-0591 Coding
25527 CATTCGAGGTAGCGCACA 174 0582-0599 Coding
25528 ACACCATCCTGTTGCAGG 175 0606-0623 Coding
25529 GAACACTTCCTTGACACC 176 0619-0636 Coding
25530 ACAGCCTCGGCGAACACT 177 0630-0647 Coding
25531 AAGAGGATGCAGGACCGC 178 0684-0701 Coding
25532 GCAGCCTCCAAGCCAAGT 179 0713-0730 3′-UTR
25533 AAAAGGCATTCAGGGAAC 180 0818-0835 3′-UTR
25534 GGGTCCAACCTTGGCTTG 181 0936-0953 3′-UTR
25535 GTCAGTAGCGGAAAATGG 182 0984-1001 3′-UTR
25536 AGCTGGATGAACTGGTCA 183 0998-1015 3′-UTR
25537 AACTGTGTGGAAAGCTGG 184 1010-1027 3′-UTR
25538 ACCACAATAGGCAGCAAC 185 1028-1045 3′-UTR
25539 GAGGGCAGAGGTTAGAGA 186 1074-1091 3′-UTR
25540 CAATTCCAAGAGCAGCGA 187 1090-1107 3′-UTR
25541 TGGAGAAGGGAGAGAGCA 188 1119-1136 3′-UTR
25542 ACATTCACCTTCTCAGGA 189 1154-1171 3′-UTR
25543 GTCAGCAAATGCGTAAGG 190 1199-1216 3′-UTR
1Emboldened residues are 2′-methoxyethoxy residues (others are 2′-deoxy-). All 2′-methoxyethoxy cytidines and cytidines are 5-methyl-cytidines; all linkages are phosphorothioate linkages.
2Co-ordinates from Genbank Accession No. X61587, locus name “HSRHOG” SEQ ID NO. 150.

RhoG inhibition data for compounds in Table 24 were obtained by real-time quantitative PCR as described in other examples herein and are averaged from three experiments. Data are shown in Table 25. If present, “N.D.” indicates “no data”.

TABLE 25
Inhibition of RhoG mRNA levels by chimeric
phosphorothioate oligonucleotides having
2′-MOE wings and a deoxy gap
SEQ
TARGET % Inhi- ID
ISIS# REGION SITE SEQUENCE bition NO.
25504 5′UTR 48 GACCTGGTGCCCCTCCCG 0 151
25505 5′UTR 73 TCTTCTGGACCCCTCTGG 32 152
25506 5′UTR 89 GGCAGTGCCTCCTCTCTC 28 153
25507 5′UTR 107 GTGCAGTTGCTGTAGTGA 0 154
25508 5′UTR 116 GCATCGTGGGTGCAGTTG 12 155
25509 Coding 137 CCACCACGCACTTGATGC 0 156
25510 Coding 185 TTGTGTAGCAGATGAGCA 0 157
25511 Coding 195 AAAGCGTTAGTTGTGTAG 33 158
25512 Coding 239 GCGCGCTGTAATTGTCGA 0 159
25513 Coding 269 GGTTCACTGTGCGCCCGT 82 160
25514 Coding 283 GTCCCACAGGTTCAGGTT 0 161
25515 Coding 319 TGTACGGAGGCGGTCATA 13 162
25516 Coding 342 ACGTTGGTCTGAGGGTAG 53 163
25517 Coding 365 CAATGGAGAAACAGATGA 0 164
25518 Coding 383 CATAGGACGGCGGACTGG 55 165
25519 Coding 393 CGCACGTTCTCATAGGAC 9 166
25520 Coding 414 ACCTCTGGATGCCACTTG 56 167
25521 Coding 430 AGGGCAGTGGTGGCACAC 0 168
25522 Coding 448 CAGCAGGATGGGCACATC 0 169
25523 Coding 488 GGGTGTCAGGCTGGGCTC 27 170
25524 Coding 537 CCCTGCTGCGGTGTGATG 55 171
25525 Coding 550 CGCGAGTGCCTGGCCCTG 41 172
25526 Coding 574 GTAGCGCACAGCGTGGAT 41 173
25527 Coding 582 CATTCGAGGTAGCGCACA 0 174
25528 Coding 606 ACACCATCCTGTTGCAGG 37 175
25529 Coding 619 GAACACTTCCTTGACACC 23 176
25530 Coding 630 ACAGCCTCGGCGAACACT 59 177
25531 Coding 684 AAGAGGATGCAGGACCGC 39 178
25532 3′UTR 713 GCAGCCTCCAAGCCAAGT 13 179
25533 3′UTR 818 AAAAGGCATTCAGGGAAC 43 180
25534 3′UTR 936 GGGTCCAACCTTGGCTTG 78 181
25535 3′UTR 984 GTCAGTAGCGGAAAATGG 54 182
25536 3′UTR 998 AGCTGGATGAACTGGTCA 54 183
25537 3′UTR 1010 AACTGTGTGGAAAGCTGG 59 184
25538 3′UTR 1028 ACCACAATAGGCAGCAAC 48 185
25539 3′UTR 1074 GAGGGCAGAGGTTAGAGA 0 188
25540 3′UTR 1090 CAATTCCAAGAGCAGCGA 26 187
25541 3′UTR 1119 TGGAGAAGGGAGAGAGCA 0 188
25542 3′UTR 1154 ACATTCACCTTCTCAGGA 26 189
25543 3′UTR 1199 GTCAGCAAATGCGTAAGG 73 190

As shown in Table 25, SEQ ID NOs 152, 158, 160, 163, 165, 167, 171, 172, 173, 175, 177, 178, 180, 181, 182, 183, 184, 185 and 190 demonstrated at least 30% inhibition of RhoG expression in this experiment and are therefore preferred.

Example 20

Human Rac1 Oligonucleotide Sequences

Antisense oligonucleotides were designed to target human Rac1. Target sequence data are from the Rac1 cDNA sequence published by Didsbury, J., et al. (J. Biol. Chem. 1989, 264, 16378-16382); Genbank accession number M29870, provided herein as SEQ ID NO: 191. Oligonucleotides were synthesized primarily with phosphorothioate linkages. Oligonucleotide sequences are shown in Table 26.

Cells were cultured, treated with oligonucleotides, and mRNA was isolated and quantitated as described in Example 2. A 45-mer antisense oligonucleotide to Rac1 (5′-ATAGAATGTGAGTCTGAACTCTTACATTTAGAACAAACAAAACCT-3′ SEQ ID NO. 192) was used as a probe as described in Didsbury, J., et al. (J. Biol. Chem. 1989, 264, 16378-16382).

An initial screen of Rac1 specific antisense oligonucleotides was performed using a oligonucleotide concentration of 300 nM.

Results are shown in Table 27. Oligonucleotides 16052 (SEQ ID NO. 195), 16056 (SEQ ID NO. 199), 16058 (SEQ ID NO. 201), 16062 (SEQ ID NO. 204) and 16063 (SEQ ID NO. 205) gave better than 50% inhibition of Rac1 mRNA levels. Oligonucleotides 16052 (SEQ ID NO. 195), 16058 (SEQ ID NO. 201) and 16062 (SEQ ID NO. 204) gave better than 75% inhibition.

TABLE 26
Nucleotide Sequences of
Rac-1 Phosphorothioate Oligonucleotides
SEQ TARGET GENE GENE
ISIS NUCLEOTIDE SEQUENCE ID NUCLEOTIDE TARGET
NO. (5′ -> 3′) NO: CO-ORDINATES1 REGION
16050 CAAATGATGCAGGACTCACA 193 0252-0271 Coding
16051 CACCACCACACACTTGATG 194 0009-0027 Coding
16052 ATAAGCCCAGATTCACCG 195 0149-0166 Coding
16053 TGTTTGCGGATAGGATAGG 196 0207-0225 Coding
16054 GCTTCTTCTCCTTCAGTTTCTC 197 0379-0400 Coding
16055 CAGCACCAATCTCCTTAGC 198 0436-0454 Coding
16056 CTCTTCCTCTTCTTCACGG 199 0542-0560 Coding
16057 CCTAAGATCAAGTTTAGTTC 200 0341-0360 Coding
16058 CGCACCTCAGGATACCACTT 201 0286-0305 Coding
16059 ATCTACCATAACATTGGCAG 202 0122-0141 Coding
16060 TAATTGTCAAAGACAGTAGG 203 0100-0119 Coding
16062 GAGCGCCGAGCACTCCAGGT 204 0461-0480 Coding
16063 GTCAAACACTGTCTTGAGGC 205 0491-0510 Coding
16143 ATAGAATGTGAGTCTGAACT 206 unknown3 3′-UTR
16144 CTTACATTTAGAACAAACAAAACCT 207 unknown3 3′-UTR
16849 CCCAGCTAAGAATTCCGCTC 208 16058 control
16850 TAAACGCCGAATCTACGC 209 16052 control
1all linkages are phosphorothioate linkages.
2Co-ordinates from Genbank Accession No. M29870, locus name “HUMRACA” SEQ ID NO. 191.
3These oligonucleotides were designed based on a probe to the 3′-UTR region of Racl (Didsbury, J., et al., J. Biol. Chem. 1989, 264, 16378-16382).

TABLE 27
Activities of Phosphorothioate Oligonucleotides Targeted to
Human Rac1
SEQ GENE
ISIS ID TARGET % mRNA % mRNA
No: NO: REGION EXPRESSION INHIBITION
LIPOFECTIN 100.0%   0.0%
only
16051 194 Coding 77.1% 22.9%
16052 195 Coding  3.7% 96.3%
16053 196 Coding 68.4% 31.6%
16054 197 Coding 67.6% 32.4%
16055 198 Coding 70.8% 29.2%
16056 199 Coding 48.0% 52.0%
16057 200 Coding 97.3%  2.7%
16058 201 Coding 22.2% 77.8%
16059 202 Coding 57.7% 42.3%
16060 203 Coding 91.6%  8.4%
16062 204 Coding 21.7% 78.3%
16063 205 Coding 32.4% 67.6%
16143 206 3′-UTR 56.1% 43.9%
16144 207 3′-UTR 72.9% 27.1%

Example 21

Dose Response and Specificity of Antisense Oligonucleotide Effects on Human Rac1 mRNA Levels in A549 Cells

Oligonucleotides 16050 (SEQ ID NO. 193), 16052 (SEQ ID No. 195), 16058 (SEQ ID NO. 201), 16062 (SEQ ID NO. 204) and 16143 SEQ ID NO. 206) were chosen for dose response studies. Oligonucleotide 16057 (SEQ ID NO. 200) was chosen as a negative control because it was inactive in the initial screen. Results are shown in Table 28. Oligonucleotides 16050, 16052, 16058 and 16062 inhibited Rac1 mRNA expression in a dose dependent manner with maximum expression of 65% to 82%.

The specificity of oligonucleotides 16052 and 16058 was tested using scrambled controls. Results are shown in Table 29. Both sequences inhibited Rac1 mRNA expression in a dose dependent manner and were significantly better than their scrambled controls.

TABLE 28
Dose Response of A549 Cells to Rac1
Antisense Oligonucleotides (ASOs)
SEQ ID ASO Gene % mRNA % mRNA
ISIS # NO: Target Dose Expression Inhibition
control LIPOFECTIN  100%   0%
only
16050 193 coding  75 nM 71.1% 28.9%
16050 193 150 nM 53.6% 46.4%
16050 193 300 nM 33.6% 66.4%
16052 195 coding  75 nM 68.2% 31.8%
16052 195 150 nM 40.5% 59.5%
16052 195 300 nM 28.3% 71.7%
16057 200 coding  75 nM 81.7% 18.3%
16057 200 150 nM 80.2% 19.8%
16057 200 300 nM 85.8% 14.2%
16058 201 coding  75 nM 60.1% 39.9%
16058 201 150 nM 42.9% 57.1%
16058 201 300 nM 17.7% 82.3%
16062 204 coding  75 nM 50.5% 49.5%
16062 204 150 nM 40.2% 59.8%
16062 204 300 nM 25.2% 74.8%
16143 206 3′-UTR  75 nM 294.8% 
16143 206 150 nM 100.8% 
16143 206 300 nM 88.6% 11.4%

TABLE 29
Specificity of Rac1 Antisense Oligonucleotides (ASOs) in
A549 Cells
SEQ ID ASO Gene % mRNA % mRNA
ISIS # NO: Target Dose Expression Inhibition
control LIPOFECTIN  100%   0%
only
16052 195 coding  75 nM 86.6% 13.4%
16052 195 150 nM 52.8% 47.2%
16052 195 300 nM 18.5% 81.5%
16850 209 control  75 nM 88.9% 11.1%
16850 209 150 nM 97.2%  2.8%
16850 209 300 nM 107.4% 
16058 201 coding  75 nM 82.7% 17.3%
16058 201 150 nM 36.8% 63.2%
16058 201 300 nM 21.1% 78.9%
16849 208 control  75 nM 90.7%  9.3%
16849 208 150 nM 70.2% 29.8%
16849 208 300 nM 68.2% 31.8%

Example 22

Design and Testing of Chimeric (Deoxy Gapped) 2′-O-methoxyethyl Rac1 Antisense Oligonucleotides on Rac1 mRNA Levels in A549 Cells

Oligonucleotides targeted to Rac1 were synthesized as a uniformly phosphorothioate or mixed phosphorothioate/phosphodiester chimeric oligonucleotides having variable regions of 2′-methoxyethyl (2′-MOE) nucleotides and deoxynucleotides. All 2′-MOE cytosines were 5-methyl-cytosines. Additionally, some oligonucleotides were synthesized with deoxycytosines as 5-methyl-cytosines. Additional oligonucleotides were synthesized, with similar chemistries, as scrambled controls. Oligonucleotide sequences and chemistries are shown in Tables 30 and 31. A dose response experiment was performed using a number of these oligonucleotides as described in Example 3.

Results are shown in Table 32. All of the chimeric oligonucleotides tested showed a dose dependent effect and showed inhibition of Rac mRNA levels comparable to that of the phosphorothioate oligodeoxynucleotide.

TABLE 30
Nucleotide Sequences of
Racl Gapmer Oligonucleotides
SEQ TARGET GENE GENE
ISIS NUCLEOTIDE SEQUENCE ID NUCLEOTIDE TARGET
NO. (5′ -> 3′) NO: CO-ORDINATES1 REGION
16899 ATAAGCCCAGATTCACCG 195 0149-0166 Coding
16900 CAAATGATGCAGGACTCACA 193 0252-0271 Coding
16901 CGCACCTCAGGATACCACTT 201 0286-0305 Coding
17161 ATAAGCCCAGATTCACCG 195 0149-0166 Coding
17162 ATAAGCCCAGATTCACCG 195 0149-0166 Coding
17163 ATAAGCCCAGATTCACCG 195 0149-0166 Coding
17164 ATAAGCCCAGATTCACCG 195 0149-0166 Coding
18540 ATAAGCCCTGATTCACCG 210 16899 mismatch
18541 ATACGCCCTGATTCACCG 211 16899 mismatch
18542 ATACGCCCTGATTAACCG 212 16899 mismatch
18549 TAAACGCCGAATCTACGC 213 16899 control
1Emboldened residues are 2′-methoxyethoxy residues (others are 2′-deoxy-). All 2′-methoxyethoxy cytidines are 5-methyl-cytidines; all linkages are phosphorothioate linkages.
2Co-ordinates from Genbank Accession No. M29870, locus name “HUMRACA” SEQ ID NO. 191.

TABLE 31
Nucleotide Sequences of Racl Mixed Backbone Oligonucleotides
SEQ TARGET GENE GENE
ISIS NUCLEOTIDE SEQUENCE ID NUCLEOTIDE TARGET
NO. (5′ -> 3′) NO: CO-ORDINATES1 REGION
17814 ToAoAoAoCoGoCoCoGsAsAsTsCsTsAsCsGsC 213 16899 control
17815 AoToAoAoGoCoCoCoAsGsAsTsTsCsAsCsCsG 195 0149-0166 Coding
17816 CoAoAoAoToGsAsTsGsCsAsGsGsAsCsToCoAoCoA 193 0252-0271 Coding
17817 AoAoAoCoToGsCsTsGsAsAsGsTsAsCsGoCoAoCoA 214 17816 control
24686 ToAoAoAoCoGoCoCoGoAoAoToCoToAoCoGoC 213 16899 control
24687 TsAsAsAsCsGsCsCsGsAsAsTsCsTsAsCsGsC 213 16899 control
1Emboldened residues are 2′-methoxyethoxy residues (others are 2′-deoxy-). All 2′-methoxyethoxy cytidines and 2′-deoxy cytidines are 5-methyl-cytidines; “s” linkages are phosphorothioate linkages, “o” linkages are phosphodiester linkages.
2Co-ordinates from Genbank Accession No. M29870, locus name “HUMRACA” SEQ ID NO. 191.

TABLE 32
Dose Response of A549 Cells to Rac1
Antisense Gapmer Oligonucleotides (ASOs)
SEQ ID ASO Gene % mRNA % mRNA
ISIS # NO: Target Dose Expression Inhibition
control LIPOFECTIN 100  0.0%
only
16899 195 coding  75 nM 79.9% 20.1%
150 nM 40.8% 59.2%
300 nM 21.8% 78.2%
17161 195 coding  75 nM 31.3% 68.7%
150 nM 16.9% 83.1%
300 nM 12.3% 87.7%
17162 195 coding  75 nM 89.2% 10.8%
150 nM 63.0% 37.0%
300 nM 18.4% 81.6%
17163 195 coding  75 nM 93.4%  6.6%
150 nM 67.3% 32.7%
300 nM 34.4% 65.6%
17164 195 coding  75 nM 94.7%  5.3%
150 nM 65.9% 34.1%
300 nM 36.2% 63.8%

Example 23

Human cdc42 Chimeric (Deoxy Gapped) 2′-O-methoxyethyl oligonucleotide Sequences

Antisense oligonucleotides were designed to target human cdc42. Target sequence data are from the cdc42 cDNA sequence published by Shinjo, K. et al. (Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 9853-9857); Genbank accession number M57298, provided herein as SEQ ID NO: 215. Oligonucleotides were synthesized as uniformly phosphorothioate chimeric oligonucleotides having a centered deoxy gap of eight nucleotides flanked by 2′-O-methoxyethyl (2′-MOE) regions. All 2′-MOE cytosines were 5-methyl-cytosines. Oligonucleotide sequences are shown in Table 33.

A549 cells were cultured and treated with oligonucleotide as described in Example 2. Quantitation of cdc42 mRNA levels was determined by real-time PCR (RT-PCR) as described in previous examples.

For cdc42 the PCR primers were:

Forward: 5′-TTCAGCAATGCAGACAATTAAGTGT-3′ SEQ ID NO. 216

Reverse: 51-TGTTGTGTAGGATATCAGGAGACATGT-3′ SEQ ID NO. 217

and the PCR probe was: FAM-TTGTGGGCGATGGTGCTGTTGGTA-TAMRA (SEQ ID NO. 218) where FAM or JOE (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.

For GAPDH the PCR primers were:

Forward primer: 5′-GAAGGTGAAGGTCGGAGTC-3′ SEQ ID NO. 65

Reverse primer: 5′-GAAGATGGTGATGGGATTTC-3′ SEQ ID NO. 66

and the PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO. 67) where FAM or JOE (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.

Results are shown in Table 34. All oligonucleotides tested gave greater than 40% inhibition of cdc42 mRNA expression.

TABLE 33
Nucleotide Sequences of cdc42 oligonucleotides
SEQ TARGET GENE GENE
ISIS NUCLEOTIDE SEQUENCE ID NUCLEOTIDE TARGET
NO. (5′ -> 3′) NO: CO-ORDINATES1 REGION
17208 TAATTGTCTGCATTGCTGAA 219 0063-0082 AUG
17209 TTACCAACAGCACCATCGCC 220 0097-0116 Coding
17210 CCACCAATCATAACTGTGAC 221 0193-0212 Coding
17211 GTGGATAACTCAGCGGTCGT 222 0270-0289 Coding
17212 GAAGATGGAGAGACCACTGA 223 0316-0335 Coding
17213 GTGAGTTATCTCAGGCACCC 224 0359-0378 Coding
17214 GCTTCTGTTTGTTCTTGGCA 225 0456-0475 Coding
17215 TGACAGCCTTCAGGTCACGG 226 0507-0526 Coding
17216 CACCTGCGGCTCTTCTTCGG 227 0613-0632 Coding
17217 TTGTCTCACACGAGTGCATG 228 0774-0793 3′-UTR
17218 TTCTGACAATACAATTACTC 229 0844-0863 3′-UTR
17219 TTACAGAGTCATCCACAAGC 230 0961-0980 3′-UTR
20457 CGATAGTCTCCACGTGAGGC 231 17215 control
21668 CGATAGTCTCCACGTGAGGC 231 17215 control
21917 GTAACGCTCCTATGGCCAGG 232 17215 control
21918 AGACTGACTGCTCGTCGCGA 233 17215 control
1Emboldened residues are 2′-methoxyethoxy residues (others are 2′-deoxy-). All 2′-methoxyethoxy cytidines are 5-methyl-cytidines, underlined “C” residues are 5-methyl-cytidines; all linkages are phosphorothioate linkages.
2Co-ordinates from Genbank Accession No. M57298, locus name “HUMGPG25K” SEQ ID NO. 215.

TABLE 34
Activities of Phosphorothioate Oligonucleotides Targeted to
Human Cdc42
SEQ GENE
ISIS ID TARGET % mRNA % mRNA
No: NO: REGION EXPRESSION INHIBITION
LIPOFECTIN  100%   0%
only
17208 219 AUG 40.6% 59.4%
17209 220 Coding 43.4% 56.6%
17210 221 Coding 55.4% 44.6%
17211 222 Coding 25.5% 74.5%
17212 223 Coding 31.1% 68.9%
17213 224 Coding 14.0% 86.0%
17214 225 Coding 27.4% 72.6%
17215 226 Coding 16.9% 83.1%
17216 227 Coding 26.0% 74.0%
17217 228 3′-UTR 28.4% 71.6%
17218 229 3′-UTR 17.2% 82.8%
17219 230 3′-UTR 20.2% 79.8%

Example 24

Dose Response of Antisense Oligonucleotide Effects cdc42 mRNA Levels in A549 Cells

Oligonucleotides 17213 (SEQ ID NO. 224), 17215 (SEQ ID No. 226), 17218 (SEQ ID NO. 229), and 17219 (SEQ ID NO. 230) were chosen for dose response studies. Results are shown in Table 35.

TABLE 35
Dose Response of A549 Cells to Cdc42
Antisense Oligonucleotides (ASOs)
SEQ ID ASO Gene % mRNA % mRNA
ISIS # NO: Target Dose Expression Inhibition
control LIPOFECTIN 100%   0%
only
17213 224 coding  75 nM 158% 
17213 300 nM 16% 84%
17215 226 coding  75 nM 90% 10%
17215 300 nM 21% 79%
17218 229 3′-UTR  75 nM 53% 47%
17218 300 nM 38% 62%
17219 230 3′-UTR  75 nM 102% 
17219 300 nM 41% 59%

Example 25

Additional cdc42 Chimeric Oligonucleotides

Oligonucleotides having SEQ ID NO: 226 were synthesized as mixed phosphorothioate/phosphodiester chimeric oligonucleotides having variable wing regions of 2′-O-methoxyethyl (2′-MOE) nucleotides and a central stretch of nine deoxynucleotides. All 2′-MOE cytosines were 5-methyl-cytosines. Oligonucleotide sequences and chemistries are shown in Table 36.

TABLE 36
Nucleotide Sequence of 17215 Analog
SEQ TARGET GENE GENE
ISIS NUCLEOTIDE SEQUENCE ID NUCLEOTIDE TARGET
NO. (51 ->31) NO: CO-ORDINATES1 REGION
22276 ToGoAoCoAoGsCsCsTsTsCsAsGsGsTsCoAoCoGoG 226 0507-0526 Coding
22277 CoGoAoToAoGsTsCsTsCsCsAsCsGsTsGoAoGoGoC 231 22276 control
1 Emboldened residues are 2′-methoxyethoxy residues (others are 2′-deoxy). All 2′-methoxyethoxy cytidines are 5-methyl-cytidines; “s” linkages are phosphorothioate linkages, “o” linkages are phosphodiester linkages.
2Co-ordinates from Genbank Accession No. M57298, locus name “HUMGPG25K” SEQ ID NO. 215.

Non-Patent Citations
Reference
1 *Branch, TIBS (Feb. 1998), 23, pp. 45-50.*
2Brenner, B., et al., "L-Selectin Regulateds Actin Polymerisation via Activation of the Small G-Protein Rac2", Biochem. Biophys. Res. Commun. 1997 231 802-07.
3delPeso, et al., "Rho proteins induce metastatic properties in vivo", Oncogene 1997 15, 3047-3057.
4Didsbury, J., et al., "rac, a Novel ras-related Family of Proteins That Are Botulinum Toxin Subsrates", J. Biol. Chem. 1989 264, 16378-26382.
5Dorseuil, O., et al., "Inhibition of Superoxide Production in B Lymphocytes by Rac Antisense Oligonucleotides",, J. Biol. Chem 1992, 267, 20540-20542.
6Engel, et al., "RhoB Is Stabilized by Transforming Growth Factor beta and Antagonizes Transcriptional Activation",J. Biol. Chem. 1998, 273, 9921-9926.
7Engel, et al., "RhoB Is Stabilized by Transforming Growth Factor β and Antagonizes Transcriptional Activation",J. Biol. Chem. 1998, 273, 9921-9926.
8 *Green et al., J. A. Coll. Surg., (Jul. 2000), vol. 191, No. 1, pp. 93-105.*
9Hall, A., "Rho GTPases and the Actin Cytoskeleton", Science, 1998 279, 509-514.
10 *Jen et al., Stem Cells 2000; 18:307-319.*
11Khosravi-Far et al., "Increasing Complexity of Ras Signal Transduction: Involvement of Rho Family Proteins", Adv. Cancer Res. 1998 72, 57-107.
12Kim, et al., "Protection from Reoxygenation Injury by Inhibition of racl", J. Clin. Invest. 1998 101, 1821-1826.
13Mellor et al., "PRK1 Is Targeted to Endosomes by the Small GTPase, RhoB", J. Biol. Chem. 1998 273, 4811-4814.
14 *Milligan et al., Journal of Medicinal Chemistry, Jul. 1993, vol. 36, No. 14, pp. 1923-1937.*
15 *Monia, Oligonucleotides As Therapeutics Agents, 1997, Wiley, Chicester (Ciba Foundation Symposium 209), pp. 107-123.*
16Narumiya et al., "rho Gene Products, Botulinum C3 Exoenzyme and Cell Adhesion", Cell Signal, 1993, 5, 9-19.
17 *Qui et al., Molecular and Cellular Biology, Jun. 1997, vol. 17, No. 6, pp. 3449-3458.*
18Ren et al., "Modulation of small G protein isoprenylation by anticancer monoterpenes in in situ mammary gland epithelial cells",Carcinogenesis 1998, 19, 827-832.
19Ridley et al., "The Small GT-Binding Protein rho Regulates the Assembly of Focal Adhesions and Actin Stress Fibers in Response to Growth Factors",Cell, 1992 70, 389-399.
20Ridley, A.J., "The GTP-binding Protein Rho", Int. J. Biochem. Cell Biol. 1997, 29, 1225-1229.
21Roux, et al., "The small GTPases Cdc42Hs, Rac1 and RhoG delineate Raf-independent pathways that cooperate to transform NIH3T3 cells", Curr. Biol. 1997 7, 629-637.
22 *Shinjo et al., Proc. Natl. Acad. Sci. USA, Dec. 1990, vol. 87, pp. 9853-9857.*
23Suwa, et al., "Overexpression of the rhoC gene correlates with progression of ductal adenocarcinoma of the pancreas", Br. J. Cancer 1998 77, 147-152.
24Takada et al., "The involvement of the rho gene product, a small molecular weight GTP-binding protein, in polyploidization of a human megakaryocytic cell line, CMK", Exp. Hemaol. 1996 24 524-530.
25Vincent, et al., "Growth-Regulated Expression of rhoG, a New Member of the ras Homolog Gene Family", Mol. Cell. Biol. 1992 12, 3138-3148.
26Vojtek, A.B., et al., "Rho Family Members: Activators of MAP Kinase Cascades", Cell 1995 82, 527-529.
27Zalcman, et al., "Regulation of Ras-related RhoB protein expression during the cell cycle", Oncogene, 1995 10, 1935-1945.
Referenced by
Citing PatentFiling datePublication dateApplicantTitle
US7772200 *Jul 21, 2006Aug 10, 2010Alnylam Pharmaceuticals, Inc.iRNA agents targeted to the Rho-A gene
US8084422Apr 4, 2008Dec 27, 2011Institut National De La Sante Et De La Recherche Medicale (Inserm)Method of treating insulin resistance with a selective inhibitor of CB2 receptor activity
US8710104Nov 6, 2009Apr 29, 2014Triact Therapeutics, Inc.Catecholic butanes and use thereof for cancer therapy
US20110251260 *Apr 7, 2011Oct 13, 2011Quark Pharmaceuticals Inc.NOVEL siRNAS AND METHODS OF USE THEREOF
CN101818150BOct 22, 2004May 16, 2012诺伊热尼有限公司Neurogenesis
EP2201982A1Dec 24, 2008Jun 30, 2010INSERM (Institut National de la Santé et de la Recherche Médicale)Histamine H4 receptor antagonists for the treatment of vestibular disorders
EP2253316A1May 20, 2009Nov 24, 2010INSERM (Institut National de la Santé et de la Recherche Medicale)serotonin 5-HT3 receptor antagonists for use in the treatment or prevention of vestibular deficits
EP2372363A1Sep 19, 2006Oct 5, 2011OSI Pharmaceuticals, Inc.Biological markers predictive of anti-cancer response to insulin-like growth factor-1
EP2399575A2Aug 8, 2007Dec 28, 2011INSERM (Institut National de la Santé et de la Recherche Medicale)Methods, uses and compositions for treatment of an infection by a virus of the family of flaviviridae through the farnesoid X receptor (FXR) inhibition
EP2399988A2Aug 8, 2007Dec 28, 2011INSERM (Institut National de la Santé et de la Recherche Medicale)Cell culture system for replication of HCV through the farnesoid X receptor (FXR) activation or inhibition and diagnostic method for HCV infection
WO2007035744A1Sep 19, 2006Mar 29, 2007Osi Pharm IncBiological markers predictive of anti-cancer response to insulin-like growth factor-1 receptor kinase inhibitors
WO2010072829A1Dec 23, 2009Jul 1, 2010INSERM (Institut National de la Santé et de la Recherche Médicale)Selective histamine h4 receptor antagonists for the treatment of vestibular disorders.
WO2010099138A2Feb 24, 2010Sep 2, 2010Osi Pharmaceuticals, Inc.Methods for the identification of agents that inhibit mesenchymal-like tumor cells or their formation
WO2010099363A1Feb 26, 2010Sep 2, 2010Osi Pharmaceuticals, Inc.Methods for the identification of agents that inhibit mesenchymal-like tumor cells or their formation
WO2010099364A2Feb 26, 2010Sep 2, 2010Osi Pharmaceuticals, Inc.Methods for the identification of agents that inhibit mesenchymal-like tumor cells or their formation
WO2010106187A2Mar 22, 2010Sep 23, 2010INSERM (Institut National de la Santé et de la Recherche Médicale)Inhibitors of cathepsin s for prevention or treatment of obesity-associated disorders
WO2010115874A1Apr 6, 2010Oct 14, 2010INSERM (Institut National de la Santé et de la Recherche Médicale)Methods for the treatment and the diagnosis ofpulmonary arterial hypertension
WO2010133663A1May 20, 2010Nov 25, 2010INSERM (Institut National de la Santé et de la Recherche Médicale)Serotonin 5-ht3 receptor antagonists for use in the treatment of lesional vestibular disorders
WO2010149765A1Jun 25, 2010Dec 29, 2010Inserm (Institut National De La Sante Et De La Recherche Medicale)Non human animal models for increased retinal vascular permeability
WO2011020874A1Aug 19, 2010Feb 24, 2011Inserm (Institut National De La Sante Et De La Recherche Medicale)Vla-4 as a biomarker for prognosis and target for therapy in duchenne muscular dystrophy
WO2011048070A1Oct 19, 2010Apr 28, 2011INSERM (Institut National de la Santé et de la Recherche Médicale)Methods and pharmaceutical compositions for the treatment of disorders of glucose homeostasis
WO2011054916A1Nov 5, 2010May 12, 2011INSERM (Institut National de la Santé et de la Recherche Médicale)Methods and pharmaceutical composition for the treatment of atherosclerosis
WO2011070049A1Dec 8, 2010Jun 16, 2011INSERM (Institut National de la Santé et de la Recherche Médicale)Endothelin inhibitors for the treatment of rapidly progressive glomerulonephritis
WO2011080261A1Dec 28, 2010Jul 7, 2011INSERM (Institut National de la Santé et de la Recherche Médicale)Method for improved cardiomyogenic differentiation of pluripotent cells
WO2011083124A1Jan 5, 2011Jul 14, 2011INSERM (Institut National de la Santé et de la Recherche Médicale)Flt3 receptor antagonists for the treatment or the prevention of pain disorders
WO2011109572A2Mar 3, 2011Sep 9, 2011OSI Pharmaceuticals, LLCBiological markers predictive of anti-cancer response to insulin-like growth factor-1 receptor kinase inhibitors
WO2011109584A2Mar 3, 2011Sep 9, 2011OSI Pharmaceuticals, LLCBiological markers predictive of anti-cancer response to insulin-like growth factor-1 receptor kinase inhibitors
WO2011141456A1May 10, 2011Nov 17, 2011INSERM (Institut National de la Santé et de la Recherche Médicale)Methods and compositions for the treatment of fluid accumulation in and/ or under the retina
WO2011151395A2Jun 1, 2011Dec 8, 2011INSERM (Institut National de la Santé et de la Recherche Médicale)Transglutaminase 2 inhibitors for use in the prevention or treatment of rapidly progressive glomerulonephritis
WO2011157798A1Jun 16, 2011Dec 22, 2011INSERM (Institut National de la Santé et de la Recherche Médicale)Methods and compositions for stimulating reepithelialisation during wound healing
WO2011163436A1 *Jun 23, 2011Dec 29, 2011Quark Pharmaceuticals, Inc.Double stranded rna compounds to rhoa and use thereof
WO2012010696A1Jul 22, 2011Jan 26, 2012INSERM (Institut National de la Santé et de la Recherche Médicale)Methods for cancer management targeting co-029
WO2012019991A1Aug 8, 2011Feb 16, 2012INSERM (Institut National de la Santé et de la Recherche Médicale)Methods and pharmaceutical compositions for the treatment of hiv-1 infections
WO2012042061A1Oct 3, 2011Apr 5, 2012INSERM (Institut National de la Santé et de la Recherche Médicale)Methods for predicting the progression and treating a chronic kidney disease in a patient
WO2012042289A1Sep 28, 2010Apr 5, 2012Inserm ( Institut National De La Sante Et De La Recherche Medicale)Methods and pharmaceutical compositions for the treatment of bone density related diseases
WO2012072820A1Dec 5, 2011Jun 7, 2012INSERM (Institut National de la Santé et de la Recherche Médicale)Methods and pharmaceutical compositions for the treatment of heart failure
WO2012107589A1Feb 13, 2012Aug 16, 2012Centre Hospitalier Universitaire D'amiensMethods and pharmaceutical compositions for the treatment and prevention of hcv infections
WO2012120130A1Mar 9, 2012Sep 13, 2012INSERM (Institut National de la Santé et de la Recherche Médicale)Methods to characterize patients suffering from hemolysis
WO2012129145A1Mar 19, 2012Sep 27, 2012OSI Pharmaceuticals, LLCNscle combination therapy
WO2012140208A1Apr 13, 2012Oct 18, 2012Inserm (Institut National De La Sante Et De La Recherche Medicale)Screening methods and pharmaceutical compositions for the treatment of inflammatory bowel diseases
WO2012146702A1Apr 27, 2012Nov 1, 2012Centre Hospitalier Universitaire De MontpellierMethods for preparing accessory cells and uses thereof for preparing activated nk cells
WO2012163848A1May 25, 2012Dec 6, 2012INSERM (Institut National de la Santé et de la Recherche Médicale)Methods and pharmaceutical compositions for the treatment of crohn's disease
WO2013014262A1Jul 27, 2012Jan 31, 2013INSERM (Institut National de la Santé et de la Recherche Médicale)Methods for diagnosing and treating myhre syndrome
WO2013024022A1Aug 10, 2012Feb 21, 2013INSERM (Institut National de la Santé et de la Recherche Médicale)Methods and pharmaceutical compositions for treatment of pulmonary hypertension
WO2013033380A1Aug 30, 2012Mar 7, 2013Genentech, Inc.Diagnostic markers
WO2013050405A1Oct 3, 2012Apr 11, 2013INSERM (Institut National de la Santé et de la Recherche Médicale)Methods and pharmaceutical compositions for the treatment of th2 mediated diseases
WO2013055530A1Sep 28, 2012Apr 18, 2013Genentech, Inc.Diagnostic methylation markers of epithelial or mesenchymal phenotype and response to egfr kinase inhibitor in tumours or tumour cells
WO2013057313A1Oct 22, 2012Apr 25, 2013INSERM (Institut National de la Santé et de la Recherche Médicale)Methods for the detection and the treatment of cardiac remodeling
WO2013068836A1Nov 6, 2012May 16, 2013INSERM (Institut National de la Santé et de la Recherche Médicale)A ddr1 antagonist or an inhibitor of ddr1 gene expression for use in the prevention or treatment of crescentic glomerulonephritis
WO2013076194A1Nov 22, 2012May 30, 2013INSERM (Institut National de la Santé et de la Recherche Médicale)Methods and pharmaceutical compositions for reducing airway hyperresponse
WO2013097940A1Dec 21, 2012Jul 4, 2013Genoplante-ValorPlants having a modulated content in seed proteins and method for production thereof
WO2013113762A1Jan 30, 2013Aug 8, 2013INSERM (Institut National de la Santé et de la Recherche Médicale)Methods and kits for predicting the risk of having a cutaneous melanoma in a subject
WO2013121034A1Feb 18, 2013Aug 22, 2013INSERM (Institut National de la Santé et de la Recherche Médicale)Methods and pharmaceutical compositions for reducing adipose tissue inflammation
WO2013156867A2Apr 18, 2013Oct 24, 2013InsermMethods and pharmaceutical compositions for the treatment of hypertension
WO2013167582A1May 7, 2013Nov 14, 2013INSERM (Institut National de la Santé et de la Recherche Médicale)Methods and pharmaceutical compositions for prevention or treatment of chronic obstructive pulmonary disease
WO2013174834A1May 22, 2013Nov 28, 2013INSERM (Institut National de la Santé et de la Recherche Médicale)Methods for diagnosing and treating focal segmental glomerulosclerosis
WO2013174997A1May 24, 2013Nov 28, 2013INSERM (Institut National de la Santé et de la Recherche Médicale)Methods and pharmaceutical compositions for the treatment of refractory haematological malignancies
WO2013182711A1Jun 10, 2013Dec 12, 2013SensorionH4 receptor inhibitors for treating tinnitus
WO2014006025A2Jul 2, 2013Jan 9, 2014INSERM (Institut National de la Santé et de la Recherche Médicale)Marker of pathogenicity in salmonella
WO2014013005A1Jul 18, 2013Jan 23, 2014INSERM (Institut National de la Santé et de la Recherche Médicale)Methods for preventing and treating chronic kidney disease (ckd)
WO2014049152A1Sep 27, 2013Apr 3, 2014INSERM (Institut National de la Santé et de la Recherche Médicale)Methods and pharmaceutical compositions for the treatment of cardiovascular fibrosis
WO2014053871A1Oct 4, 2012Apr 10, 2014INSERM (Institut National de la Santé et de la Recherche Médicale)A method for screening a compound capable of inhibiting the notch1 transcriptional activity
WO2014057045A1Oct 10, 2013Apr 17, 2014INSERM (Institut National de la Santé et de la Recherche Médicale)Methods and pharmaceutical compositions for treatment of gastrointestinal stromal tumors
WO2014064203A1Oct 24, 2013May 1, 2014INSERM (Institut National de la Santé et de la Recherche Médicale)Lyve-1 antagonists for preventing or treating a pathological condition associated with lymphangiogenesis
WO2014064215A1Oct 24, 2013May 1, 2014INSERM (Institut National de la Santé et de la Recherche Médicale)TPL2 KINASE INHIBITORS FOR PREVENTING OR TREATING DIABETES AND FOR PROMOTING β-CELL SURVIVAL
WO2014072416A1Nov 7, 2013May 15, 2014INSERM (Institut National de la Santé et de la Recherche Médicale)Methods and pharmaceutical compositions for the treatment of bone metastases
WO2014076235A1Nov 15, 2013May 22, 2014NeurochloreModulators of intracellular chloride concentration for treating fragile x syndrome
WO2014170435A2Apr 17, 2014Oct 23, 2014INSERM (Institut National de la Santé et de la Recherche Médicale)Methods and pharmaceutical compositions for inhibiting lymphocyte proliferation in a subject in need thereof
WO2014170712A1Apr 15, 2013Oct 23, 2014INSERM (Institut National de la Santé et de la Recherche Médicale)Rac-1 inhibitors or pi3k inhibitors for preventing intestinal barrier dysfunction
Classifications
U.S. Classification435/375, 435/91.1, 435/325, 536/23.1, 536/24.31, 435/366, 536/24.5, 435/6.14
International ClassificationC12Q1/68, C07H21/00, A61K38/00, C12N15/113
Cooperative ClassificationC12N2310/346, C12N2310/321, C12N2310/316, C12N15/1137, A61K38/00, C12Y301/05001, C12N2310/334, C12N2310/315, C07B2200/11, C12N2310/341, C12N2310/322, C12N2310/345, C12N2310/3341, C12N2310/318, C07H21/00, C12N2310/3183, C12N2310/335
European ClassificationC07H21/00, C12Y301/05001, C12N15/113D
Legal Events
DateCodeEventDescription
Aug 12, 2014FPExpired due to failure to pay maintenance fee
Effective date: 20140625
Jun 25, 2014LAPSLapse for failure to pay maintenance fees
Jan 31, 2014REMIMaintenance fee reminder mailed
Nov 20, 2009FPAYFee payment
Year of fee payment: 8
Nov 23, 2005FPAYFee payment
Year of fee payment: 4
Jan 7, 2000ASAssignment
Owner name: ISIS PHRAMACEUTICALS INC., CALIFORNIA
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:ROBERTS, M. LUISA;COWSERT, LEX M.;REEL/FRAME:010519/0935
Effective date: 19991222
Owner name: ISIS PHRAMACEUTICALS INC. CARLSBAD RESEARCH CENTER